Preparation and characterization of rod-like chitosan–quinoline nanoparticles as pH-responsive nanocarriers for quercetin delivery

Article abstract of DOI:10.1016/j.ijbiomac.2019.01.137

Novel chitosan–quinoline nanoparticles as anticancer drug nanocarriers were prepared using 2-chloro-3-formylquinoline and 3-formylquinolin-2(1H)-one as non-toxic modifying agents via oil–in–water nanoemulsion technique. Chitosan–quinoline nanoparticles were characterized by FT–IR, UV–vis spectrophotometry, XRD, SEM, AFM and DLS techniques. The morphological and particle size studies demonstrated that drug–loaded chitosan–quinoline nanoparticles have a regular nanorod shape and monolithic structure with the desired particle size of 141 to 174.8 nm and a negative zeta potential of ?2.4 to ?14.1 mV. Drug loading capacity (LC) and encapsulation efficiency (EE) were achieved using quercetin as a hydrophobic anticancer drug and were about 4.8–9.6% and 65.8–77%, respectively. The in vitro release studies displayed great pH-sensitive release behavior. Evaluation of the anticancer efficacy of quercetin loaded chitosan–quinoline nanoparticles using the in vitro cytotoxicity studies against HeLa cells indicated that the chitosan nanoparticles are a promising candidate for the anticancer drugs delivery.

Full text of DOI:10.1016/j.ijbiomac.2019.01.137

Accepted Manuscript
Preparation and characterization of rod-like chitosan–quinoline
nanoparticles as pH-responsive nanocarriers for quercetin
delivery
Shahnaz Rahimi, Sepideh Khoee, Mehdi Ghandi
PII:
S0141-8130(18)35290-5
DOI:
https://doi.org/10.1016/j.ijbiomac.2019.01.137
BIOMAC 11570
Reference:
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
4 October 2018
10 January 2019
24 January 2019
Please cite this article as: S. Rahimi, S. Khoee and M. Ghandi, Preparation and
characterization of rod-like chitosan–quinoline nanoparticles as pH-responsive
nanocarriers for quercetin delivery, International Journal of Biological Macromolecules,
https://doi.org/10.1016/j.ijbiomac.2019.01.137
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
Preparation and characterization of rod-like chitosan–quinoline
nanoparticles as pH-responsive nanocarriers for quercetin delivery
Shahnaz Rahimi,
School of Chemistry, College of Science, University of Tehran, Tehran, Iran
sh.rh1989@gmail.com
Sepideh Khoee*,
School of Chemistry, College of Science, University of Tehran, , PO Box 14155 6455,
Tehran, Iran, Tel: (098) 21 61113301, Fax
Khoee@khayam.ut.ac.ir
Mehdi Ghandi,
School of Chemistry, College of Science, University of Tehran, Tehran, Iran
ghandi@khayam.ut.ac.ir
*
Corresponding author
1

ACCEPTED MANUSCRIPT
Abstract
Novel chitosan–quinoline nanoparticles as anticancer drug nanocarriers were prepared using
-chloro-3-formylquinoline and 3-formylquinolin-2(1H)-one as non-toxic modifying agents
2
via oil–in–water nanoemulsion technique. Chitosan–quinoline nanoparticles were
characterized by FT–IR, UV–vis spectrophotometry, XRD, SEM, AFM and DLS techniques.
The morphological and particle size studies demonstrated that drug–loaded chitosan–
quinoline nanoparticles have a regular nanorod shape and monolithic structure with the
desired particle size of 141 to 174.8 nm and a negative zeta potential of −2.4 to −14.1 mV.
Drug loading capacity (LC) and encapsulation efficiency (EE) were achieved using quercetin
as a hydrophobic anticancer drug and were about 4.8–9.6% and 65.8–77%, respectively. The
in vitro release studies displayed great pH-sensitive release behavior. Evaluation of the
anticancer efficacy of quercetin loaded chitosan–quinoline nanoparticles using the in vitro
cytotoxicity studies against HeLa cells indicated that the chitosan nanoparticles are a
promising candidate for the anticancer drugs delivery.
Keywords: Chitosan; 2-chloro-3-formylquinoline; 3-formylquinolin-2(1H)-one; nanorod
shape; drug delivery system.
2

ACCEPTED MANUSCRIPT
1
. Introduction
Chitosan is a natural biopolymer generally produced by deacetylation of chitin with
excellent accessibility, biodegradability, biocompatibility, nontoxicity and antimicrobial
activity [1]. Chitosan has been extensively used as a material in numerous biomedical and
pharmaceutical fields, such as gene delivery, biosensors, tissue engineering and drug delivery
systems. Chitosan and its derivatives have received the most attention among researchers
owing to its various physicochemical features and biological activities [2-6]. Due to its
excellent adhesion property, chitosan can enhance the cell membrane permeability [7].
Accordingly, various drug carriers are designed using chitosan and its derivatives. Fu et al.
developed chitosan hollow microspheres (CHM) as a new carrier for tough hydrophobic
drugs based on an interfacial Schiff-base bonding reaction [8]. Liu et al. designed
monodisperse core-shell chitosan microcapsules via a crosslinking reaction of O/W/O double
emulsion of chitosan and terephthalaldehyde, which exhibited a pH-responsive burst release
of hydrophobic drugs [9]. These findings show that chitosan and its derivatives are suitable
candidates for drug carriers. However, concerns about the safety of chitosan particles remains
owing to the toxicity of organic crosslinking agents especially glutaraldehyde, which causes
adverse effects on the human body [10,11]. Natural and non-toxic products possessing
biological properties can be used as crosslinking agents to overcome the potential side
effects. Therefore, the choice of the appropriate crosslinking agent is a novel and interesting
challenge for the preparation of chitosan nanoparticles [12,13].
Quinolines are important biological compounds because of their occurrence in a large
number of natural products especially in alkaloids, and their broad range of applications in
pharmaceuticals, medicine and agrochemicals [14,15]. Due to their good efficacy and
desirable safety profiles, compounds containing quinoline have attracted considerable
attention for their diverse bioactivities like antifungal [16], anti-inflammatory [17,18],
3

ACCEPTED MANUSCRIPT
antimalarial [19], antiviral [20], antimicrobial [21,22], anticancer [23,24] and analgesic
activities [18]. Moreover, quinoline and its derivatives have been widely utilized in medicinal
chemistry due to the occurrence of their structure in the number of commercial drugs such as
mefloquine [25], quinine [26], amodiaquine [27] and chloroquine [28]. Among the quinoline
derivatives, 2,3-disubstituted quinolines and 3-substituted quinolin-2-ones have played an
important role in the design and development of novel compounds with marvelous
anticancer activities [29,30]. Inspired by the known properties of quinoline and its
derivatives, we undertook a study for the synthesis of novel drug–loaded chitosan–quinoline
nanoparticles with 2-chloro-3-formylquinoline (CFQ) and 3-formylquinolin-2(1H)-one
(FQO) as non-toxic modifying agents via oil–in–water (O/W) nanoemulsion method. The
obtained chitosan–quinoline nanoparticles were characterized by means of FT–IR, XRD,
UV–vis, SEM imaging techniques and other spectroscopic methods. Quercetin was loaded
into the modified chitosan nanoparticles as a model anticancer drug. Furthermore, the release
of quercetin from the chitosan–quinoline nanoparticles was evaluated and cytotoxicity against
HeLa cells was also investigated using MTT assay.
2
. Experimental
2
.1 Materials
Chitosan (CS) with low molecular weight (MW = 50,000–190,000 Da, degree of
deacetylation: 85%), Dulbecco's Modified Eagle's medium (DMEM) and 3-(4,5-
dimethylazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-
Aldrich. Quercetin was purchased from Fluka Chemical Co. Phosphoryl chloride (POCl3),
N,N-dimethylformamide (DMF), acetanilide, glacial acetic acid (CH COOH, 100%), Tween
3
6
0 (polyoxyethylene sorbitan monostearate, HLB = 14.9), dichloromethane (CH Cl , 99%),
2 2
ethanol (EtOH, 99%) and other commercially available chemicals were supplied from Merck
Chemical Company and used without further purification.
4

ACCEPTED MANUSCRIPT
2
.2 Measurements
1
The H NMR of the organic products was recorded on a Bruker 500 MHz NMR
spectrometer using CDCl as the solvent at 25 °C. Melting points were carried out by an
3
Electrothermal model 9100 apparatus and are uncorrected. Mass spectra of the organic
compounds were obtained using an HP (Agilent technologies) 5937 Mass Selective Detector.
Fourier transform infrared (FT–IR) spectra were performed in KBr pellets by FT–IR
-1
spectrophotometer (IR Affinity, Shimadzu, Japan) using KBr in the range of 600–4000 cm .
The average particle size and zeta potential of the chitosan–quinoline nanoparticles were
determined by dynamic light scattering (DLS, Brookhaven instrument, USA) at 25 °C in
triplicate. Each sample was diluted to the desired concentration using deionized water and the
analysis was carried out at a fixed scattering angle of 90°. Ultraviolet–visible (UV–vis)
absorption spectra were determined on a UV-VIS spectrophotometer (Jasco V750, Jasco,
Tokyo, Japan). The shape of the obtained nanoparticles was analyzed by scanning electron
microscopy (FE–SEM) (HITACHI S–4160, Japan). Samples were mounted on an aluminium
stub using a double adhesive carbon tape and then sputter–coated with gold before
observations. X–ray diffraction (XRD) analysis of the samples was conducted by an X–ray
diffractometer (Rigaku, Japan) with CuKα radiation as target. The measurement was carried
out at a voltage of 40 kV and 40 mA current and 2θ angle range from 5° to 50° at a scanning
-1
speed of 4 min at room temperature. The topography of the drug–loaded chitosan–
quinoline nanoparticles was characterized by atomic force microscopy (AFM, ENTEGRA
AFMNT–MDT, China) on a freshly cleaved mica substrate.
2
.3 Synthesis and characterization of 2-chloro-3-formylquinoline
Dry DMF (2.7 mL, 34.65 mmol) was cooled to 0–5 ºC in a round bottom flask, and
POCl (9 mL, 98.28 mmol) was added dropwise by a dropping funnel to DMF with stirring.
3
After stirring the solution for about 15 min, acetanilide (1.4 g, 10.37 mmol) was added and
5

ACCEPTED MANUSCRIPT
the mixture heated at 75–80 °C for 8 h. The reaction mixture was then poured into crushed
ice under vigorous stirring for about 30 minutes. The precipitate thus appeared was filtered,
washed well with cold water and dried. Recrystallization of the crude compound from ethyl
acetate finally afforded the product 1 in high yield (90 %), mp: 147–148 °C; FT–IR (KBr), ν,
−
1
1
cm : 1682 (HC=O), 943 (C–Cl); H NMR (500MHz, CDCl ): δ 7.61 (t, J = 7.3 Hz, 1H),
3
7
.86 (t, J = 7.3 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 8.1 Hz, 1H), 8.72 (s, 1H), 10.52
+
(
s, 1H); ESI-MS m/z: [M+H] calcd. for C H ClNO = 191.01; found 191.3 (Figure S1).
1
0
6
2
.4 Synthesis and characterization of 3-formylquinolin-2(1H)-one
A suspension of 2-chloro-3-formylquinoline (1 mmol) in acetic acid (70%, 10 mL) was
stirred under reflux for 4–6 h. After completion as indicated by TLC, the reaction mixture
was cooled to room temperature and the precipitated product filtered, washed with water and
−
1
dried. (yield 93%; mp: 302–303 °C); FT–IR (KBr), ν, cm : 3153 (N–H), 1666 (HC=O),
1
1
1
1
623 NHC=O); H NMR (500MHz, CDCl ): δ 7.72 (t, J = 7.1 Hz, 1H), 7.93 (t, J = 7.1 Hz,
3
H), 8.05 (d, J = 8.3 Hz, 1H), 8.14 (d, J = 8.3 Hz, 1H), 8.76 (s, 1H), 10.27–10.48 (br s, 1H),
+
0.63 (s, 1H); ESI-MS m/z: [M+H] calcd. for C H NO = 173.1; found 173.0 (Figure S2).
1
0
7
2
2
.5 Preparation of chitosan-quinoline nanoparticles
Chitosan–quinoline nanoparticles were prepared via the O/W nanoemulsion system.
Briefly, chitosan (1%, w/v) was dissolved in dilute acetic acid solution (0.7%, v/v) at ambient
temperature and vigorously stirred overnight (1400 rpm). After addition of Tween 60 (1%,
w/v), the mixture was sonicated in an ultrasonic probe for 15 min until the aqueous phase
mixture became homogeneous. Quercetin was dissolved in EtOH-CH Cl solution (1:3, v/v)
2
2
to obtain a final concentration of 10% (w/w of chitosan) and stirred for 10 min until the oil
phase mixture became transparent. Thereafter, the oil phase was added dropwise into the
aqueous phase and sonicated for 5 min to form O/W emulsion. The volume ratio of O/W was
6

ACCEPTED MANUSCRIPT
fixed at 1:5. Afterwards, a solution of quinoline derivatives (0.5%, w/v) as the modifying
agents was poured into the O/W nanoemulsion dropwise to prepare the crosslinked chitosan–
quinoline nanoparticles suspension. The crosslinked chitosan nanoparticles solution was then
centrifuged at 16,000 rpm for 30 min at 20 °C. Finally, the obtained chitosan–quinoline
nanoparticles were washed well with deionized water and freeze–dried for 12 h. The blank
chitosan–quinoline nanoparticles prepared similarly without adding of quercetin.
2
.6 Drug loading and encapsulation efficiency
The encapsulation efficiency (EE) and loading capacity (LC) of quercetin loaded in
chitosan–quinoline nanoparticles were determined by centrifugation of the drug–loaded
chitosan–quinoline nanoparticles at 16,000 rpm for 30 min to remove the non–entrapped
quercetin. The clear supernatant was analyzed to measure the ultraviolet absorbance by using
a UV–vis spectrophotometer at 373 nm. EE and LC were thus estimated from Eqs. (1) and
(2), respectively:
ꢀ
ꢁꢂ ꢀꢃ
EE (%) =
ꢄꢅꢆꢇꢇ
(1)
(2)
ꢀ
ꢁ
ꢀ
ꢁꢂ ꢀꢃ
ꢈꢉꢅꢊꢋꢌꢍꢅꢅ
ꢄꢅꢆꢇꢇ
ꢎꢏꢐꢑꢒꢁꢅꢓꢃꢅꢔꢕꢔꢓꢖꢕꢗꢁꢐꢘꢙꢏꢚ
Where QCt is the total amount of quercetin used in the preparation of nanoparticles and
QCf is the free quercetin present in the supernatant.
2
.7 In vitro quercetin release
The quercetin release profile of the chitosan–quinoline nanoparticles was carried by a
dialysis method. Typically, the weighed freeze–dried quercetin–loaded chitosan–quinoline
nanoparticles were dispersed in release medium (phosphate buffer saline (PBS), pH 7.4 and
pH 5.8) with a concentration of 1 mg/mL at a membrane dialysis bag (cut off 12,000 kDa).
7

ACCEPTED MANUSCRIPT
The end–sealed dialysis bag was suspended into a container with 10 mL of PBS at the same
pH value as that in the bag. The outer phase of the release media was maintained at 37 ± 0.5
°
C with continuous stirring at a speed of 50 rpm. At prescheduled time intervals, 5 mL of
samples were withdrawn in both of pH medium and replaced with an equal volume of fresh
media to maintain a constant volume. The cumulative amount of quercetin released from
chitosan–quinoline nanoparticles in each buffer was determined by measuring the absorbance
at 373 nm in a UV–vis Spectrophotometer. To identify the mechanism for the release of
quercetin from crosslinked and non-crosslinked chitosans, the suitability of Higuchi [31] and
Korsmeyer‒Peppas [32] equations were evaluated according to the following equations
(Equations 3 and 4, respectively):
ꢛꢜ
ꢍꢞꢟꢠ
(3)
(4)
ꢛꢝ
ꢡꢠ
ꢣ ꢤ
ꢍꢞꢠ
ꢡꢢ
Where, Mt is cumulative amounts of released drug at time t and M∞ is cumulative amounts
of released drug at infinite time. K and k' are Higuchi and Korsmeyer‒Peppas constant. In the
case of rod shape polymeric vehicles, n values that is used as release mechanism
characterization are listed in Table 1.
Table 1. Diffusion exponent and solute release mechanism for rod shape matrices
Diffusion exponent (n)
n<0.45
Overall solut diffusion mechanism
Fickian diffusion
0
.43<n<0.89
Anomalous (non- Fickian) diffusion
Case II transport
0
.89<n<1
n>1
Super case II transport
8

ACCEPTED MANUSCRIPT
2
.8 In vitro cytotoxicity evaluation
MTT assay using HeLa cell lines was employed for determination of the in vitro
cytotoxicity of free quercetin and the quercetin–loaded chitosan–quinoline nanoparticles.
4
Typically, HeLa cells were seeded into 96–well plates at the density of 1.25×10 cells per
well in 180 μL DMEM and then incubated in humidified incubator of 5% CO at 37 ℃for 24
2
h. After that, the medium was replaced by fresh corresponding medium containing solutions
of pure quercetin and drug–loaded chitosan–quinoline nanoparticles at concentrations of 10
−
1
−1
to 400 μg mL . After incubated for 48 h, 20 μL of MTT solution (5 μg mL in PBS) was
added to every well and the cells were incubated for another 4 h. The medium containing
MTT was then removed, and displaced by 150 μL of dimethyl sulphoxide (DMSO) per well
for 10 min at ambient temperature to dissolve the formazan crystals. Finally, the absorbance
of the solution was measured at a wavelength of 570 nm using a microplate reader and the
relative cell viability (%) was calculated by the following equation:
ꢬꢭꢚꢅꢊꢁꢏꢚꢁꢅꢘꢏꢙꢙꢌ
ꢉꢥꢦꢦꢅꢧꢨꢩꢪꢨꢦꢨꢠꢫꢅꢊꢋꢌꢍꢅꢅ
ꢄꢅꢆꢇꢇꢋ (3)
ꢬꢭꢚꢅꢊꢘꢓꢔꢁꢗꢓꢙꢙꢏꢮꢅꢘꢏꢙꢙꢌ
2
.9. Optical Microscopy Analysis
4
HeLa cells were cultured in 24-well plates at a density of 1 × 10 cells per well. After 24 h of
incubation, cells were incubated without nanoparticles as control or with different
concentration (100, 200 and 400 g/mL) of two types of chitosan nanorods for 4 h at 37 °C.
After that, morphologies of the cells were observed using an optical microscope.
3
. Results and discussion
3
.1 Design, synthesis and characterization
9

ACCEPTED MANUSCRIPT
CFQ and FQO were prepared according to the procedure previously reported [30,33] as
the modifying agents for the preparation of chitosan–quinoline nanoparticles. The synthetic
routes are presented in Scheme 1. CFQ was synthesized starting from acetanilide via a
Vilsmeier–Haack reaction. Subsequently, heating CFQ in aqueous acetic acid at reflux led to
1
FQO in good yield (93%). All spectral data including IR, H NMR and Mass of 2-chloro-3-
formylquinoline and 3-formylquinolin-2(1H)-one were consistent with those of authentic
samples.
Scheme 1. Synthesis of CFQ e and FQO
1
The H NMR spectrum of CFQ revealed characteristic signals at δ 10.52 (s, 1H) and
7
.61-8.04 (m, 5H) due to the aldehyde and aromatic protons, respectively (Figure 1A).
1
0

ACCEPTED MANUSCRIPT
1
Figure 1. (A) H-NMR spectra of CFQ and (B) FT-IR spectra of CFQ (a) and FQO (b)
Similarly, the appearance of signals at δ 10.27-10.48 (brs, 1H), 10.63 (s, 1H) and 7.72-
1
8
.76 (m, 5H) due to the NH, aldehyde and aromatic protons in the H NMR spectrum of FQO
is consistent with the suggested structure (Figure S3). The IR spectrum of CFQ exhibited the
1
1

ACCEPTED MANUSCRIPT
-1
-1
main signals at 1682 cm and 941 cm for C=O and C–Cl, respectively. The most prominent
signals in the IR spectrum of FQO corresponded to the intense absorption bands at 3153 and
-1
1
666–1623 cm attributed to the amide NH and carbonyl groups together with the
-1
disappearance of signal at 941 cm for C–Cl absorption (Figure 1B).
The chitosan–quinoline nanoparticles were prepared by Schiff base reaction of the
chitosan chains amine and the quinoline aldehyde groups (Scheme 2). Moreover, the
chitosan–quinoline nanoparticles are further stabilized through the formation of hydrogen
bonding between FQO amide groups. This in turn enhances the physicochemical properties
of chitosan–quinoline network. On the other hand, because of the biological activity of
quinoline compounds and their non-toxicity properties, it is expected that the incorporation of
quinoline into chitosan polymer furnishes novel nanoparticles with modified biological
activity.
1
2

ACCEPTED MANUSCRIPT
Scheme 2. Synthesis of chitosan–quinoline nanoparticles modified with CFQ (A) and with FQO (B)
The FT–IR spectra of pure chitosan and chitosan–quinoline nanoparticles are presented
in Figure 2. The pure chitosan spectrum reveals the absorption peaks in 3200–3600, 3425
-1
and 2873 cm due to the intra– and intermolecular different –OH hydrogen bonds, N–H/O–
H, and C–H stretching vibrations, respectively. Whereas other characteristic peaks of
−
1
chitosan appears at 1654, 1592 and 1375 cm are assigned to the amide I C=O, amide II NH
1
3

ACCEPTED MANUSCRIPT
and amide III NHCO stretching vibrations, respectively, the broad peak displayed at 1025
-1
cm was attributed to the C–O absorption [34,35].
Figure 2. FT-IR spectra of the pure chitosan (A), blank chitosan–quinoline nanoparticles (B) and drug-loaded
chitosan–quinoline nanoparticles crosslinked with CFQ (C) and FQO (D)
Compared to the pure chitosan, the strong absorption peaks observed at 1682 and 1670
-1
cm in the spectrum of the chitosan–quinoline nanoparticles due to C=N stretching vibrations
indicate that imine bond formation has occurs via a chemical modification between the
1
4

ACCEPTED MANUSCRIPT
chitosan chains amine and quinoline aldehyde groups [36,37]. Other specific peaks
-1
displayed at 752, 815, 1451 and 1612 cm are attributed to the quinoline aromatic ring.
-1
Finally, observation of an increase in the absorptions at 2864 and 2918 cm concomitant with
-1
the omission of absorbance at 2000–3900 cm is significant, perhaps due the formation of
chitosan–quinoline nanoparticles.
The XRD spectrum of pure chitosan presented in Figure 3A revealed the two broad
peaks at 2θ = 12 and 20° in the former and in agreement with the previous reports [38]. In the
XRD patterns of drug–loaded chitosan–quinoline nanoparticles, diffractive region is observed
at 2θ of 16, 22, 29, 36, 39, 43, 47, and 48°. Observation of higher intensity in the XRD
pattern of drug–loaded chitosan nanoparticles crosslinked with quinoline derivatives indicates
their more crystalline structure. As such, it can be concluded that chemical modification with
quinoline derivatives has converted the amorphous chitosan nanoparticles into a crystalline
form.
The UV–vis absorption spectra of pure chitosan and chitosan–quinoline particles at 25
°
C are shown in Figure 3B. Whereas the UV–vis absorption spectrum of pure chitosan is
transparent and shows only a weak absorption band at 224 nm, those for chitosan–quinoline
particles indicate two maxima at 250 and 306 nm due to the existence of π → π* and n → π*
transitions of the azomethine chromophore moiety and quinoline heterocyclic ring. In
addition, the UV–vis absorption spectrum of chitosan particles crosslinked with FQO
exhibited a broad absorption band between the 360–450 nm regions, which could be assigned
to the amide delocalized π-bond.
1
5

ACCEPTED MANUSCRIPT
Figure 3. A: XRD patterns of chitosan powder (a), drug–loaded chitosan nanoparticles crosslinked with FQO
(
b) and drug–loaded chitosan nanoparticles crosslinked with CFQ (c). B: UV-Vis spectra of the pure chitosan
(
a), chitosan nanoparticles crosslinked with FQO (b) and chitosan nanoparticles crosslinked with CFQ (c).
The particle size of the chitosan–quinoline nanoparticles was determined by dynamic
light scattering (DLS) technique. Because of the adhesion property of chitosan in aqueous
solution, the nanoparticles tend to produce aggregates, which lead to an increase in their
1
6

ACCEPTED MANUSCRIPT
average hydrodynamic diameter [39]. The progressive dispersion of the nanoparticles in
aqueous suspension has been suggested as an efficient method of decreasing the inter–particle
interactions [40] and utilized in this research. As indicated in Figure 4A and B, the average
diameter of 150.7 nm with a zeta potential of −2.4 mV of blank chitosan nanoparticles
crosslinked with CFQ changes to 174.8 nm with a zeta potential of −10.8 mV upon loading of
quercetin. Accordingly, the average diameter of 141.2 nm with a zeta potential of −5.7 mV of
the blank chitosan nanoparticles crosslinked with FQO also changes to 165.1 nm with a zeta
potential of −14.1 mV upon loading of quercetin (Figure 4C and D). As such, the suitability
of the chitosan–quinoline nanoparticles with negative zeta potential values as a drug delivery
system is concluded.
Figure 4. DLS results of chitosan nanoparticles crosslinked with CFQ (A) and FQO (C), and their zeta potential
results: CFQ (B) and FQO (D)
1
7

ACCEPTED MANUSCRIPT
The characterization of morphology and topography of the chitosan–quinoline
nanoparticles was carried out using SEM and AFM techniques. The SEM images of blank
and quercetin–loaded chitosan nanoparticles crosslinked with quinoline derivatives presented
in Figure 5 reveals that the blank chitosan–quinoline nanoparticles have spherical shape and
nanosize structure, also reveal nearly uniform distribution with no intense particle
agglomeration.
Figure 5. SEM images of the blank chitosan nanoparticles crosslinked with CFQ (A), crosslinked with FQO
(
B), drug-loaded chitosan nanoparticles crosslinked with CFQ (upper surface (C) and side surface (D)) and
crosslinked with FQO (E) and (F)
1
8

ACCEPTED MANUSCRIPT
The morphology of quercetin–loaded chitosan nanoparticles crosslinked with CFQ is a
monolithic structure with a kind of pattern and the morphology of quercetin–loaded chitosan
nanoparticles crosslinked with FQO is nanorod structure along with uniformity in size
(Figure 5C-F). Differences in the morphology of the blank chitosan–quinoline nanoparticles
and quercetin–loaded chitosan–quinoline nanoparticles can be attributed to the intermolecular
hydrogen bonding and π–π (π–stacking) interactions present between quercetin and quinoline
derivatives (Scheme S1).
In addition, AFM topographic images and 3D models of the surface topography
(Figure 6) confirmed the nanorod-like morphology and nearly homogenous structure of
quercetin–loaded chitosan nanoparticles made by crosslinking with quinoline derivatives, in
agreement with SEM observations. The rod–like nanoparticles exhibited the enhanced cell
adhesion, improved cell proliferation and transfection of living cells via their higher surface
areas, with more effective penetration in tumors in comparison to those of spherical
analogues [41]. Therefore, the quercetin–loaded chitosan nanoparticles crosslinked with
quinoline derivatives having such surface morphology is favored for use in biomedical
applications, especially drug delivery systems.
1
9

ACCEPTED MANUSCRIPT
Figure 6. AFM images (2D) and (3D) of drug-loaded chitosan nanoparticles crosslinked with CFQ (A) and
drug-loaded chitosan nanoparticles crosslinked with FQO (B)
3
.2 Drug loading capacity and in vitro release profile
Quercetin, a potent and hydrophobic anticancer drug, was employed to measure the
drug loading and release profile of the chitosan–quinoline nanoparticles. Low encapsulation
efficiency was not expected due to the hydrophobic and π–π (π–stacking) interactions as well
as intermolecular hydrogen bonding present between quercetin and the crosslinked chitosan
nanoparticle quinoline groups. Accordingly, the drug LC and EE of the chitosan
nanoparticles crosslinked with FQO determined as 9.6% and 77.2%, respectively, were
higher than those of 4.8% and 65.8% found for chitosan nanoparticles crosslinked with CFQ.
These efficiencies are higher than that found for quercetin-loaded chitosan nanoparticles
modified with glycyrrhetinic acid [42]. The encapsulation efficiency depends on several
parameters and one of the most important ones is the strong affinity between hydrophobic
2
0

ACCEPTED MANUSCRIPT
drug and hydrophobic domains in nanocarrier. It was found that this hydrophobic domains in
chitosan could be obtained by adding hydrophobic co-materials to the carrier formulation,
like lecithin [43], or pre-adhering of quercetin to carriers before particle crosslinking [44]. In
this report, we used the former method, by inserting aromatic crosslinkers in the structure of
chitosan. Consequently, a larger amount of quercetin preferred to encapsulate in nanocarriers
during nanoparticle formation process. However, these interactions may reduce the diffusion
of quercetin from nanocarriers during the release process. On the other hand, the low
solubility of quercetin in water, automatically forced the drug molecules to attract to the
surface or outer layers of nanoparticle and enhance its encapsulation efficiency. But,
afterward, a complete release of drug in short period of time is observable in these systems.
These results exhibit the improved stability of the quercetin–loaded chitosan nanoparticles
crosslinked with FQO. The quercetin release profile of the chitosan–quinoline nanoparticles
and chitosan nanoparticles without using quinoline derivatives in response to pH values of
7
.4 and 5.8 was studied in a dialysis setup at 37 °C. Based on the in vitro release profile, it
was found that the chitosan nanoparticles crosslinked with quinoline derivatives release
quercetin much faster at pH 5.8 than that of pH 7.4. In acidic environment at low pH value
(pH < 6), the amino groups of chitosan are protonated and positively charged, so its
intramolecular electrostatic repulsion and hydrophilicity enhancement make chitosan
nanoparticles swell dramatically. By protonation of chitosan amino groups, the Schiff base
bonding becomes instable and decomposition of the crosslinkers occurred. Therefore, by
using such a reversible crosslinking reaction between chitosan and 2-chloro-3-
formylquinoline and 3-formylquinolin-2(1H)-one, an interesting pH-responsive controlled
release process can be achieved for acid-triggered burst release with the proposed
microcapsule. As shown in Figure 7, the burst release of chitosan nanoparticles crosslinked
with FQO and CFQ is 69.3% and 78.4%, at pH 5.8 during the first 8 h, respectively. The
2
1

ACCEPTED MANUSCRIPT
quercetin release mechanism at this step could be illustrated by the quercetin diffusion
localized at the surface of the chitosan–quinoline nanoparticles [45,46]. On the other hand,
the cumulative release of chitosan–quinoline and chitosan–quinolone nanoparticles at pH 7.4
is 56.4% and 52.7% in first 8 h, respectively. After 150 h, whereas the drug release of
chitosan nanoparticles crosslinked with CFQ was approximately completed at pH 5.8, only
8
2% of drug release occurred from chitosan nanoparticles crosslinked with FQO under
similar condition. This could be explained by the stronger intermolecular hydrogen bonding
between quercetin and FQO, which caused the lower release rate in comparison with chitosan
nanoparticles crosslinked with CFQ.
2
2

ACCEPTED MANUSCRIPT
Figure 7. In vitro release of quercetin from drug-loaded chitosan nanoparticles in PBS under different pH
conditions (The inset shows the results for the first 10 hours.)
Mechanism of drug release from crosslinked and non-crosslinked chitosan were analyzed at
two pHs by Higuchi and Korsmeyer-peppas equations and their data were listed in Table 2.
2
Higuchi’s release rate constant (k) and its correlation coefficient values (r ) were determined.
The release data was also fitted into Korsmeyer‒Peppas model and by considering release
rate constants (k') and release constants (n), the fitting accuracy was calculated using
2
correlation coefficient values (r ). When comparing these two model, the best fit method is
Korsmeyer‒Peppas, regarding to highest values of correlation coefficients, accordingly, the
release mechanisms for all samples at both pHs are Fickian diffusion.
Table 2. Estimated values obtained by fitting the drug release data to the Higuchi and
Korsmeyer-Peppas models
Higuchi
Korsmeyer-peppas
Chitosan nanorods
pH
Release mechanism
2
2
k
r
k'
r
n
Crosslinked with FQO
Crosslinked with CFQ 7.4 0.1735 0.7668 0.7974 0.9045 0.2060
Non-crosslinked
Crosslinked with FQO
Crosslinked with CFQ 5.8 0.2116 0.9338 0.7334 0.9980 0.2598
Non-crosslinked 0.1916 0.9470 0.7741 0.9816 0.2306
0.1866 0.8156 0.8109 0.9076 0.2213
Fickian diffusion
Fickian diffusion
Fickian diffusion
Fickian diffusion
Fickian diffusion
Fickian diffusion
0.1610 0.8795 0.8073 0.9546 0.1887
0.2017 0.9763 0.5040 0.9957 0.2568
The SEM images of quercetin–loaded chitosan nanoparticles crosslinked with quinoline
derivatives at pH 7.4 and 5.8 presented in Figure 8. In neutral medium (pH 7.4), the
quercetin–loaded chitosan nanoparticles crosslinked with quinoline derivatives maintain good
nanorod shape and structural integrity, which promises that quercetin localized at the matrix
of the chitosan–quinoline nanoparticles would not be released before reaching the tumor
cells. While, in acidic medium (pH 5.8), the quercetin–loaded chitosan–quinoline
nanoparticles have lost their nanorod shape and converted into tiny nanocubic crystals and
2
3

ACCEPTED MANUSCRIPT
consequently their disposal of the body is more easily. With the protonation of the chitosan
amine groups at low pH, the imine bonds between the chitosan chains and quinoline
molecules becomes weaken and instable, which led to decomposition of the crosslinked
chitosan nanoparticles and quercetin was eventually released very rapidly. Therefore, by
utilization of such a reversible crosslinking reaction between chitosan and quinoline
derivatives, an interesting pH-sensitive controlled release system can be achieved.
Figure 8. SEM images of quercetin-loaded chitosan nanoparticles crosslinked with CFQ in pH 7.4 and 5.8 (A)
and (B) respectively, quercetin-loaded chitosan nanoparticles crosslinked with FQO
3
.3 Cell viability assays
MTT assay was used to evaluate the in vitro cytotoxicity of quercetin–loaded chitosan–
quinoline nanoparticles, quercetin–loaded chitosan nanoparticles without of modification
with quinoline derivatives, CFQ, FQO and the free quercetin at different concentrations from
−
1
1
0 to 400 μg mL , using HeLa cells as model. All the formulations exhibited remarkable
2
4

ACCEPTED MANUSCRIPT
anticancer activity against HeLa cells after 48 h incubation. As shown in Figure 9, as the
concentration of quercetin increased, the cell viability was further reduced. The half maximal
inhibitory concentration (IC ) value of the chitosan nanoparticles crosslinked with CFQ and
5
0
−
1
FQO and free quercetin against HeLa cell lines were 10, 14 and about 32 μg mL ,
respectively. These results showed that quercetin–loaded chitosan–quinoline nanoparticles
reveal higher cytotoxicity for cancer cell proliferation in comparison to that of free quercetin
and quercetin–loaded chitosan nanoparticles without of modification with quinoline
derivatives. Significantly, no remarkable difference was observed in cell viability between
the quercetin–loaded chitosan nanoparticles crosslinked with quinoline derivatives during the
experiment. Therefore, it can be concluded that chitosan–quinoline nanoparticles are able to
enter the cells and show favorable pharmacological effect on cancer cells. Beside the
anticancer activity of drug-loaded chitosan nanorods with two crosslinkers, the higher cell
viability of non-crosslinked sample despite its higher drug release is interesting. In fact, after
internalization of crosslinked nanoparticles by cancer cells, in the acidic media of lysosome
and endosome the imine linkages of quinoline crosslinkers were quickly cleaved to
protonated amino groups. This positive charge encourages nanoparticles to
endosome/lysosome escape, which in turn provides the opportunity to increase system
circulation efficiency and enhance favorable cellular and subcellular bioavailability [47].
2
5

ACCEPTED MANUSCRIPT
Figure 9. Cell viability (%) of HeLa cancer cells after incubation with different quercetin formulations for 48 h
Optical microscopy was employed to examine the influences of different concentration of
two kinds of crosslinked chitosan nanoparticles on the morphology of HeLa cells. It can be
seen from Figure 10, that the cells which were incubated with drug-loaded nanorods even
after 4 hours incubation could change the spindle-like morphology of untreated cell (control
cells) to rounded one. On the other hand, the images of HeLa cells incubated with higher
concentrations of NPs show an increase in internalization, the presence of nanoparticles
2
6

ACCEPTED MANUSCRIPT
inside the cells is the evidence of this phenomena. A homogeneous distribution of NPs inside
the cells is also noteworthy.
Figure 10. Optical microscopy images of HeLa cells incubated with different concentrations of chitosan
crosslinked with quinoline derivatives: before treatment and after 4h of treatment
4
. Conclusions
In summary, novel rod–like drug–loaded chitosan–quinoline nanoparticles were
successfully designed and synthesized via an oil–in–water nanoemulsion method, using 2-
chloro-3-formylquinoline and 3-formylquinolin-2(1H)-one as friendly and non-toxic
crosslinking agents. The structures of the obtained nanoparticles were carefully studied. The
2
7

ACCEPTED MANUSCRIPT
chitosan–quinoline nanoparticles were used as nanocarriers for efficient encapsulation of
quercetin, exposing a pH–sensitive controllable release. The nanoparticles showed particle
size in the range of 141–174 nm. The morphology of quercetin–loaded chitosan nanoparticles
crosslinked with CFQ and FQO was a monolithic structure with a kind of pattern and a
regular nanorod shape, respectively. Due to the cleavage of crosslinked imine linkages
present between chitosan and quinoline derivatives, the in vitro results displayed an
accelerated drug release at lower pH condition in comparison to that occur under
physiological conditions. Moreover, the in vitro quercetin release data showed that the
chitosan nanoparticles crosslinked with FQO displayed a slow drug release in comparison to
that of chitosan nanoparticles crosslinked with CFQ. In addition, the SEM images of
quercetin–loaded chitosan nanoparticles crosslinked with quinoline derivatives in acidic
medium exhibited that nanoparticles have lost their nanorod shape and converted into tiny
nanocubic crystals, which led to decomposition of the crosslinked chitosan nanoparticles and
quercetin was eventually released very rapidly. Compared to free quercetin, the quercetin–
loaded chitosan–quinoline nanoparticles showed impressive comparable cytotoxicity against
HeLa cells. Based on the obtained results, utilization of this new type of chitosan
nanoparticles crosslinked with quinoline derivatives as potential drug delivery systems is
concluded.
References
[
1] M.R. Kumar, R.A. Muzzarelli, C. Muzzarelli, H. Sashiwa, A. Domb, Chitosan chemistry and
pharmaceutical perspectives, Chemical reviews 104 (2004) 6017-6084.
2
8

ACCEPTED MANUSCRIPT
[
[
2] D. Depan, P.V. Surya, B. Girase, R. Misra, Organic/inorganic hybrid network structure
nanocomposite scaffolds based on grafted chitosan for tissue engineering, Acta biomaterialia 7
(
2011) 2163-2175.
3] M. Diaconu, S.C. Litescu, G.L. Radu, Laccase–MWCNT–chitosan biosensor—A new tool for
total polyphenolic content evaluation from in vitro cultivated plants, Sensors and Actuators B:
Chemical 145 (2010) 800-806.
[
[
4] R. Jayakumar, D. Menon, K. Manzoor, S. Nair, H. Tamura, Biomedical applications of chitin
and chitosan based nanomaterials—A short review, Carbohydrate Polymers 82 (2010) 227-232.
5] S.F. Peng, M.J. Yang, C.J. Su, H.L. Chen, P.W. Lee, M.C. Wei, H.W. Sung, Effects of
incorporation of poly (γ-glutamic acid) in chitosan/DNA complex nanoparticles on cellular
uptake and transfection efficiency, Biomaterials 30 (2009) 1797-1808.
[
6] Y.Q. Ye, F.L. Yang, F.Q. Hu, Y.Z. Du, H. Yuan, H.Y. Yu, Core-modified chitosan-based
polymeric micelles for controlled release of doxorubicin, International journal of pharmaceutics
352 (2008) 294-301.
[
[
[
7] M. Thanou, J.C. Verhoef, H.E. Junginger, Oral drug absorption enhancement by chitosan and
its derivatives, Advanced drug delivery reviews 52 (2001) 117-126.
8] Y.N. Fu, Y. Li, G. Li, L. Yang, Q. Yuan, L. Tao, X. Wang, Adaptive Chitosan Hollow
Microspheres as Efficient Drug Carrier, Biomacromolecules 18 (2017) 2195-2204.
9] L. Liu, J.P. Yang, X.J. Ju, R. Xie, Y.M. Liu, W. Wang, J.J. Zhang, C.H. Niu, L.Y. Chu,
Monodisperse core-shell chitosan microcapsules for pH-responsive burst release of
hydrophobic drugs, Soft Matter 7 (2011) 4821-4827.
[
[
10] K. Gupta, F.H. Jabrail, Glutaraldehyde and glyoxal cross-linked chitosan microspheres for
controlled delivery of centchroman, Carbohydrate research 341 (2006) 744-756.
11] A.P. Rokhade, N.B. Shelke, S.A. Patil, T.M. Aminabhavi, Novel interpenetrating polymer
network microspheres of chitosan and methylcellulose for controlled release of theophylline,
Carbohydrate Polymers 69 (2007) 678-687.
2
9