Design, synthesis, characterization and biological evaluation of Thieno[2,3?b]pyridines?chitosan nanocomposites as drug delivery systems for colon targeting

Article abstract of DOI:10.1016/j.carres.2020.107990

Thieno[2,3?b]pyridine derivatives DATP_a?c have been synthesized based on Thorpe?Ziegler Cyclization. The reaction of arylidene malononitrile derivatives (I_a?c) with thiocyanoacetamide (II) in basic medium (piperidine) followed by alkylation using ethyl chloroacetate and finally, cyclization in sodium ethoxide yielded DATP_a?c. Thieno[2,3?b]pyridine?chitosan nanocomposites CS?DATP_a?c were prepared from the DATP_a?c and CS nanoparticles using sodium tripolyphosphate (TPP). CS?DATP_a?c nanocomposites were characterized using FTIR, TEM and XRD techniques and showed a relatively narrow size distribution of monodispersed nanoparticles with the average size of 14–78 nm. The in vitro release studies of CS?DAΤP_a?c nanocomposites were investigated and showed that the drug release rate is pH-dependent and the trend is as follows: basic > neutral > acidic. The faster release rate in basic medium effectively prolongs drug delivery in gastric pH. Additionally, the antibacterial investigation showed that DATP_a?c and CS?DATP_a?c nanocomposites exhibited antibacterial activity against both Gram-positive and Gram-negative bacteria but CS?DATP_a?c nanocomposites showed much higher antibacterial activity compared to the DATP_a?c, which in agreement with the particle size measurements as DATP_a?c are in the bulky structure whereas, CS?DATP_a?c are in the nanostructure. The results may have applications of drug design for colon targeting.

Full text of DOI:10.1016/j.carres.2020.107990

CarbohydrateResearch492(2020)107990
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Design, synthesis, characterization and biological evaluation of Thieno[2,
3−b]pyridines−chitosan nanocomposites as drug delivery systems for
colon targeting
Hanaa Mansour∗, Ahmed I. Khodair, Samia M. Elsiginy, Amal E. Elghanam
Department of Chemistry, Faculty of Science, Kafrelsheikh University, Kafrelsheikh, 33516, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Thieno[2,3−b]pyridine derivatives DATPa−c have been synthesized based on Thorpe−Ziegler Cyclization. The
reaction of arylidene malononitrile derivatives (Ia−c) with thiocyanoacetamide (II) in basic medium (piperidine)
Thienopyridines
Chitosan
followed by alkylation using ethyl chloroacetate and finally, cyclization in sodium ethoxide yielded DATPa−c
.
Nanocomposites
Drug release
Antibacterial
Colon targeting
Thieno[2,3−b]pyridine−chitosan nanocomposites CS−DATPa−c were prepared from the DATPa−c and CS
nanoparticles using sodium tripolyphosphate (TPP). CS−DATPa−c nanocomposites were characterized using
FTIR, TEM and XRD techniques and showed a relatively narrow size distribution of monodispersed nanoparticles
with the average size of 14–78 nm. The in vitro release studies of CS−DAΤPa−c nanocomposites were in-
vestigated and showed that the drug release rate is pH-dependent and the trend is as follows: basic
>
neutral > acidic. The faster release rate in basic medium effectively prolongs drug delivery in gastric pH.
Additionally, the antibacterial investigation showed that DATPa−c and CS−DATPa−c nanocomposites exhibited
antibacterial activity against both Gram-positive and Gram-negative bacteria but CS−DATPa−c nanocomposites
showed much higher antibacterial activity compared to the DATPa−c, which in agreement with the particle size
measurements as DATPa−c are in the bulky structure whereas, CS−DATPa−c are in the nanostructure. The
results may have applications of drug design for colon targeting.
1. Introduction
especially in the treatment of adverse inflammatory, autoimmune,
cardiovascular, proliferative and nociceptive conditions [20]. Haya-
Chitosan (CS), a biopolymer, has been largely studied as a phar-
maceutical excipient for colon-specific delivery [1,2]. The advantages
of CS are biocompatibility, nontoxicity, biodegradability, antibacterial
activity, low cost and ease of chemical modifications. It has been ex-
amined extensively in the pharmaceutical industry for the development
of a drug delivery system [3]. CS nanoparticles are used as drug carriers
and showed controlled drug release, however, the low water-solubility
under physiological pH was the main disadvantage [4]. To overcome
this limitation, many composite/nanocomposite of chitosan with other
compounds have been designed and investigated and showed new
properties via the interaction of the constituents [5,6].
kawa et al. have evaluated the functional groups connected to the
central fused ring and their positions in terms of biological activity
[21–23]. They found that the cytotoxicity of compound A decreased
when the amino group was masked with one methyl group (B) (Fig. 1).
On the other hand, the cytotoxicity had completely disappeared when
the amino group was masked with two methyl groups. These results
revealed that the functional groups contributed mainly to the biological
activity and the central fused ring and its substituents are important. In
this study, we have inserted another amine in addition to other sub-
stituents in the benzene ring and also inserted ethyl ester moiety instead
of the phenyl ketone moiety (DATPa−c Scheme 1) to investigate the
relationship between the structure and biological activity of the deri-
vatives. Also, the solubility and the accessibility are important factors
for controlling the reactivity of any drug and thus thieno[2,3−b]pyr-
idine derivatives DATPa−c (Scheme 1) were loaded on chitosan nano-
Thieno[2,3−b]pyridines, reported for the first time in 1913 [7], are
important fused heterocyclic compounds [8] and have a broad spec-
trum of biological activities [9]. Some of them possess cytotoxic activity
[10–14] anti-inflammatory [14,15] antiviral [16] or antibacterial
[17,18] activity. They act as hypolipoproteinemic and antiathero-
sclerotic agents [19] and are of benefit as pharmaceutical agents,
particles
to
give
the
thienopyridines−CS
nanocomposite
(CS−DATPa−c). The in vitro release studies of CS−DATPa−c
∗ Corresponding author.
E-mail addresses: mansourhanaa@yahoo.com, hanaa.mansour@sci.kfs.edu.eg (H. Mansour).
https://doi.org/10.1016/j.carres.2020.107990
Received 11 February 2020; Received in revised form 10 March 2020; Accepted 23 March 2020
Availableonline29March2020
0008-6215/©2020ElsevierLtd.Allrightsreserved.

H. Mansour, et al.
CarbohydrateResearch492(2020)107990
Fig. 1. a) FT−IR (KBr), b) 1H NMR (DMSO−d6) and c) 13C NMR (DMSO−d6) spectra of DATPc.
Scheme 1. Schematic representation of thieno[2,3−b]pyridine derivatives A, B, C and DATPa−c
.
nanocomposites were investigated using the sonication method and
UV–vis spectroscopy to investigate their pharmaceutical activities. The
preliminary antibacterial activities of DATPa−c and CS−DATPa−c
against Escherichia coli, Salmonella choleraesuis, Staphylococcus aureus,
and Proteus mirabilis bacteria were tested in vitro and discussed.
spectrophotometer Cambridge, England. X−ray diffraction patterns
(XRD) were obtained by Shimadzu 6000 instrument equipped with a Cu
anode, automatic divergence slit and a graphite monochromator, under
the following experimental conditions: CuKa radiation, 1.54 °A; gen-
erator tension, 45 kV; generator current, 40 mA; intensity ratio (a2/a1),
0.500. The morphology of the samples was examined using a trans-
mission electron microscope (TEM) JEOL: JEM-100cx) to determine the
particle size and particle shape. 1H NMR spectra were obtained using
Bruker DPX 400 MHz spectrometer with DMSO−d6 as solvent and TMS
as an internal reference. The UV−VIS spectra were obtained using
PerkinElmer UV/VIS Lambda 2 spectrometer. Mass spectra were carried
out using EI mode on Direct Inlet part to the mass analyzer in Thermo
Scientific GCMS model ISQ at the Regional Center for Mycology and
Biotechnology (RCMB), Al−Azhar University, Nasr City, Cairo.
2. Experimental part
2.1. Instrumentations
All reagents and solvents were used as purchased without further
purification. Analytical TLC was performed using silica gel 60 F254
plates (Merck). Fourier transform infrared (FT−IR) spectra were ob-
tained in the transmission mode using Mattson 1000, Unicam infrared
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H. Mansour, et al.
CarbohydrateResearch492(2020)107990
2.2. Syntheses
2H, NH2); 7.20–7.40 (m, 4H, Ar–H); 13C NMR (DMSO−d6):
δ
(ppm)
= 14,9 (CH3); 56.5 (OCH3); 60.2 (CH2); 116.2 (CN);
2.2.1. Preparation of 6−Amino−3,5−dicyano−4−(p−nitrophenyl)
pyridin(1H)−2−thiones (IIIc)
125.6–160.8 (Ar–C); 165.3 (C]O).
A mixture of arylidene malononitrile Ic (0.01 mol) and thiocya-
noacetamide II (0.01 mol) in ethanol (50 ml) was refluxed in the pre-
sence of catalytic amounts of piperidine for 3 h. The solvent then was
removed and the solid product, formed after the addition of diluted
hydrochloric acid, was collected by filtration, washed with water and
then recrystallized from ethanol. IR(KBr), cm−1: 3350,3206 (NH2);
2222 (CN); 1241(C]S); 1H NMR (DMSO‑d6): δ (ppm) = 7.60 (br, 2H,
NH2); 13.02 (br,1H, NH); 7.60–8.30 (m, 4H, Ar–H); 13C NMR
(DMSO‑d6): δ (ppm) = 114.2,114.7 (CN); 123.0–157.3 (Ar–C); 197.8
(C]S).
2.2.3.3. 3,6−diamino−4−(p−nitrophenyl)thieno−{2,3−b}pyridines
(DATPc). IR(KBr), cm−1
: 3501, 3430, 3366 (NH2); 2215(CN);
1651(C]O); 1H NMR (DMSO‑d6): δ (ppm) = 1.22–1.28 (t, 3H, CH3);
4.17–4.22 (q, 2H, CH2); 5.50 (s, 2H, NH2 exchanged by D2O); 7.50 (s,
2H, NH2 exchanged by D2O); 7.80–8.40 (m, 4H, Ar–H); 13C NMR
(DMSO−d6):
δ (ppm) = 14,60 (CH3); 60.4 (CH2); 115.8 (CN);
124.7–159.1 (Ar–C); 165.4 (C]O).
2.2.4. Synthesis of CS−DATPa−c nanocomposites
Thienopyridine derivatives DATPa−c (0.0011 mol) was added to CS
solution (1 g in 80 ml of 1% acetic acid) with stirring for 30 min. Then
TPP (sodium tripolyphosphate) (1 g in 5 ml of water) was added during
sonication for 15 min followed by stirring for 2 h. The products were
separated using a centrifuge and dried at 40 °C.
2.2.2. General procedure for the preparation of compounds IVa−c
Pyridinethiones (0.01 mol) was stirred at room temperature in DMF
(7 ml) and KOH (10%, 2.7 ml) for 1 h. To this solution, ethyl chlor-
oacetate (0.02 mol) was added dropwise with keeping the temperature
at 25 °C. The reaction was monitored using TLC and at the end of the
reaction, H2O (5 ml) was added. The solid product formed was filtered
2.3. In vitro drug release
off and washed with ethanol to give IVa−c
.
The release study was carried out as follows: About 50 mg the
sample was added to the buffer (50 ml) and the system was maintained
at 37 °C throughout the study. A certain amount of the release medium
was collected at appropriate intervals and the amount of drug was
detected using a UV spectrometer at 378 nm. The percentage of drug
release was calculated according to Equation (1).
2.2.2.1. Ethyl
2−(6−amino−3,5−dicyano−4−phenylpyridin−2−thiolyl)acetate
(IVa). IR(KBr), cm−1: 3465, 3353 (NH2); 2204 (CN); 1723(C=Oester);
1H NMR (DMSO‑d6): δ (ppm) = 1.22–1.25 (t, 3H, CH3); 4.10 (s, 2H,
CH2); 4.15–4.20 (q, 2H, CH2); 7.56–7.59 (m, 5H, Ar–H); 8.00 (s, 2H,
NH2 exchanged by D2O); 13C NMR (DMSO−d6): δ (ppm) = 14.5 (CH3);
32.4 (CH2CO); 61.8 (CH2); 115.5, 115.6 (CN); 128.4–166.0 (Ar–C);
168.5 (C]O).
Drug released (%) = (At/A∞) x 100
(1)
Where At and A∞ are the absorbances of releasing the drug at time t and
the absorbance of a completely releasing drug, respectively.
2.2.2.2. Ethyl
2−(6−amino−3,5−dicyano−4−(p-methoxyphenyl)
pyridin−2−thiolyl)acetate (IVb). IR(KBr), cm−1: 3417, 3306 (NH2)
2213(CN); 1731(C]O); 1H NMR (DMSO‑d6): δ (ppm) = 1.21–1.25 (t,
3H, CH3); 3.80 (s, 3H, OCH3); 4.10 (s, 2H, CH2); 4.13–4.19 (q, 2H,
CH2); 7.10–7.50 (m, 4H, Ar–H); 7.90 (s, 2H, NH2 exchanged by D2O);
13C NMR (DMSO‑d6): δ (ppm) = 14.5 (CH3); 32.4 (CH2CO); 55.8
(OCH3); 61.8 (CH2); 115.5, 115.6 (CN); 126.2–165.9 (Ar–C); 168.5 (C]
O).
2.4. Antibacterial activity
The groups of bacteria Escherichia coli, Pseudomonas aeuroguinsa
(Gram-negative bacteria) and staphylococcus aureus, Enterococcus fae-
calis (Gram-positive bacteria) were used to investigate the antibacterial
activities of the thienopyridines and their nanocomposites. Cut plug
method, recorded by Pridham et al. 1956 [24], was used as follows:
Freshly prepared spore suspension of different test microorganisms
(0.5 ml of about 106 cells/ml) was mixed with 9.5 ml of nutrient agar
medium (for bacteria) at 45 °C, poured on sterile Petri dishes, and left to
solidify at room temperature. Regular wells were made in the in-
oculated agar plates by a sterile cork borer of 0.7 mm diameter. Each
well was filled with 20 mg of each tested powder. Three replicas were
made for each test, and all plates were incubated at 32 °C for 24 h for
bacteria. Then the average diameters of inhibition zones were recorded
in centimeters and compared for all plates. MIC (minimal inhibitory
concentration) was measured as follows: Half-fold serial dilutions were
made for selected compounds to prepare concentrations of 10–70 mg/
ml in distilled water and zero concentration was considered as a ne-
gative control. A previously prepared pure spore suspension of each test
microorganism (0.5 ml of about 106 cells/ml) was mixed with 9.5 ml of
each concentration in sterile test tubes, incubated at 32 °C for 24 h for
bacteria, then optical density of growth was measured by spectro-
photometer (Optima SP-300, Japan) at 620 nm for each incubated
mixture, results were represented graphically, and MIC was recorded
for each tested material [25].
2.2.2.3. Ethyl 2−(6-amino−3,5−dicyano−4−(p−nitrophenyl)pyridin−
2−thiolyl)acetate (IVc). IR(KBr), cm−1: 3446, 3316 (NH2), 2214(CN),
1742(C]O); 1H NMR (DMSO‑d6): δ (ppm) = 1.22–1.25 (t, 3H, CH3);
4.10 (s, 2H, CH2); 4.17–4.33 (q, 2H, CH2); 7.80–8.40 (m, 4H, Ar–H); 8.10
(s, 2H, NH2 exchanged by D2O); 13C NMR (DMSO‑d6): δ (ppm) = 14.4
(CH3); 32.4 (CH2CO); 61.9 (CH2); 115.2, 115.3 (2CN); 124.4–166.1
(Ar–C); 168.4 (C]O).
2.2.3. General procedure for the thienopyridines DATPa−c
Compound IVa−c (0.003 mol) was refluxed in sodium ethoxide
(0.1 N) and the reaction was monitored by TLC. The solid product
formed was collected by filtration and washed with ethanol.
2.2.3.1. 3,6−diamino-4-phenylthieno−{2,3−b}pyridines (DATPa). IR
(KBr), cm−1: 3501, 3353, 3297(NH2); 2214 (CN); 1686 (C]O); 1H
NMR (DMSO‑d6): δ (ppm) = 1.23–1.26 (t, 3H, CH3); 4.17–4.22 (q, 2H,
CH2); 5.50 (s, 2H, NH2 exchanged by D2O); 7.50–7.54 (m, 5H, Ar–H);
7.60 (s, 2H, NH2 exchanged by D2O); 13C NMR (DMSO‑d6):
δ
(ppm) = 14,9 (CH3); 60.3 (CH2); 116.0 (CN); 128.4–159.2 (Ar–C);
165.3 (C]O).
3. Results and discussion
2.2.3.2. 3,6−diamino-4-(p-methoxyphenyl)thieno−{2,3−b}pyridines
(DATPb). IR(KBr), cm−1: 3491, 3353, 3232 (NH2); 2213 (CN); 1666
(C]O); 1H NMR (DMSO−d6): (ppm) = 1.22–1.26 (t, 3H, CH3);
4.16–4.21 (q, 2H, CH2); 5.60 (s, 2H, NH2, exchanged by D2O); 7.1 (s,
3.1. Synthesis and characterization of thienopyridine derivatives DATPa−c
According to the pathway shown in Scheme 2, a stepwise of
synthesis thienopyridines DATPa−c was carried out under mild reaction
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H. Mansour, et al.
CarbohydrateResearch492(2020)107990
6−amino−3,5−dicyano−4−aryl−pyridine−2(1H)−thione deriva-
tives (IIIa−c) were obtained in a high percentage yield. Different
spectroscopic techniques were used to confirm the formation of IIIa−c
and the structures of compounds IIIa and IIIb are similar to that re-
ported previously [28]. IR absorption spectrum of IIIc shows char-
acteristic bands at 3350, 3266, 2222 and 1241 cm−1 assigned for NH2,
NH, cyano and C]S groups, respectively. The 1H NMR spectrum of IIIc
exhibits bands at δ = 7.60 and 13.02 ppm assigned for the NH2 and NH
protons (exchanged by D2O). The aromatic protons appear at
δ = 7.60–8.30 ppm. The 13C NMR spectrum of IIIc shows two bands at
δ = 114.2 and 114.7 ppm assigned for the two cyano groups. The C]S
group appears at δ = 197.8 ppm, whereas, the aromatic carbons appear
at
δ
=
123.0–157.3. 2−Alkylthio−3,5−dicyanoaminopyridines
(IVa−c
) were obtained by alkylation of 3,5−dicyanoaminopyr-
idine−2(1H)−thiones (IIIa−c
) using ethyl chloroacetate in basic
medium.
The structure of the isolated 2−alkylthio−3,5−dicyano-amino-
pyridines (IVa−c) was confirmed by different analytical methods. IR
spectrum of IVb shows the appearance of a new band at 1731 cm−1
assigned for COester. The NH2 group appears at 3417 and 3306 cm−1
,
while the cyano groups appear as a sharp band at 2213 cm−1
.
1H NMR
Scheme 2. Synthesis of the thienopyridine derivatives DATPa-c; i) EtOH, pi-
(DMSO-d6) spectrum of IVb exhibits a triplet at δ = 1.21–1.25 ppm and
a multiplet at δ = 4.13–4.19 ppm assigned for the CH3 and CH2 protons
of the ethyl group, respectively. Two singlet bands appear at δ = 3.80
and 4.10 ppm assigned for the OCH3 and S–CH2 protons, respectively.
The aromatic protons appear as a multiplet at δ = 7.10–7.50 ppm. The
amine protons appear as a singlet band at δ = 7.90 ppm (exchanged by
peridine, ii) ClCH2COOEt, KOH, DMF; iii) Sodium ethoxide.
conditions based on Thorpe-Ziegler Cyclization [26]. Firstly, and ac-
cording to Knoevenagel procedure [27], the reaction of arylidene
malononitrile derivatives (Ia−c
) with thiocyanoacetamide (II) in
ethanol
in
the
presence
of
a
base
(piperidine),
Fig. 2. TEM micrographs of CS−DATP nanocomposites; a) CS−DATPa, b) CS−DATPb; c) CS−DAΤPc; and d) CS nanoparticle.
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H. Mansour, et al.
CarbohydrateResearch492(2020)107990
Fig. 3. XRD patterns of DATPa−c, CS−DATPa−c nanocomposites, chitosan and chitosan nanoparticles.
are generated by cyclization of 2-alkylthio-3,5-dicyano-aminopyridines
(IVa-c) in sodium ethoxide under reflux conditions. The structure of
DATPa−c is confirmed by IR, NMR, UV/Vis spectroscopy, mass spec-
trometry, and elemental analysis. IR spectrum of DATPb (Fig. 1a) shows
the C=Oester at 1666 cm−1 which shows a large shift compared to the
corresponding IVb(1731 cm−1), indicating for a new thieno ring for-
mation. The amino groups appear at 3494, 3353 and 3232 cm−1
whereas; the cyano group appears as only one at 2213 cm−1. The 1H
NMR spectrum of DATPb (Fig. 1b) exhibits
a
triplet at
δ = 1.22–1.26 ppm and a quartet at δ = 4.16–4.21 ppm, assigned for
the CH3 and CH2 of the ester group. A new band appears as singlet band
δ = 5.60 ppm for the newly formed amino protons (exchanged by D2O),
while the other amino appears at δ = 7.10 ppm (exchanged by D2O).
The aromatic protons appear as multiplet band at δ = 7.20–7.40 ppm.
The 13C NMR spectrum of DATPb (Fig. 1c) is characteristic by the ap-
pearance of only one cyano carbon at δ = 116.2 ppm, while the other
cyano group disappeared via the cyclization process. The ethyl ester
carbons appear at δ = 14.9 and 60.3 ppm. The carbonyl carbon of the
ester group is found at δ = 165.3 ppm. The methylene protons at
δ = 32.4 ppm are disappeared via the new ring formation. The aro-
matic carbons are found at δ = 125.6–160.8 ppm. The UV/Vis spec-
trum of DATPc is distinguished by the appearance of bands at
Fig. 4. FT-IR spectra of CS nanoparticles, DATPb and CS−DATPb nano-
composite.
λ
max = 327 nm. The EI−MS spectra of DATPa-c showed the molecular
D2O), which is shifted to a higher field compared to the pyridine thione
IIIb. 13C NMR spectrum confirmed the structure of IVb which is dis-
tinguished by the disappearance of the C]S band at δ = 188.5 ppm
and the appearance of a new band at δ = 168.5 ppm characteristic for
the C=Oester. The CH3 and CH2 carbons of the ester group appear at
δ = 14.5 and 61.8 ppm respectively. A new band at δ = 32.4 ppm is
characteristic for the (CH2CO) group. The other bands have no dis-
tinguished changes as the (OCH3) group appears at δ = 55.8 ppm, the
two cyano groups found at δ = 115.5 and 115.6 ppm and the aromatic
carbons are found at δ = 126.2–165.9 ppm. Thienopyridine DATPa-c
ion peak, indicating the product formation.
3.2. Preparation and characterization of CS−DATPa-c nanocomposites
The morphology of CS−DATPa-c nanocomposites was characterized
by the TEM technique. The TEM image, depicted in Fig. 2, shows a
relatively narrow size distribution of monodispersed nanopartiThe
surface morphology characteristicscles with an average size of
14–78 nm. The spherical shape is due to the loading of the
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CarbohydrateResearch492(2020)107990
Table 1
Zeta potential (mV) of DATPa−c and CS−DATPa−c nanocomposites.
DATPa
DATPb
DATPc
−12.2
CS−DATPa
−0.8
CS−DATPb
+4.8
CS−DATPc
−9.4
Compound
Zeta potential (mV)
−22.5
−27.5
thienopyridine derivatives with a deviation of 5 nm [29,30] have an
impact on bioadhesion. It has been found that the nanospheres with a
coarser and more porous surface may offer enhanced bioadhesivity as
compared to those with a smoother texture.
chitosan nanoparticles and this is in agreement with other results due to
the encapsulation of thienopyridines in the polymer networks [32].
The FT−IR spectra of DATPb, CS−DATPb nanocomposites, and CS
nanoparticles were recorded to observe the difference in structure due
to DATP incorporation into the matrix (Fig. 4). The main characteristic
peaks of the chitosan spectrum were: peaks at 3511−3491 cm−1 as-
signed to –O−H stretching; peaks at 3353−3374 cm−1 assigned to
NH− and NH2− stretching and the peaks at 1639 cm−1 assigned for
the amide stretching. Typical signals for saccharide structures appeared
between 1205 and 900 cm−1 [33]. After the loading process, the IR
spectra of CS−DATPa−c showed some differences between the na-
nochitosan and those containing DATP. Especially, bands around
3490 cm−1, typical for νOH and νNH became broader and shifted to
about 3510 cm−1. This indicated that the interaction is via the imino
group of the chitosan moiety. Another feature showing the successful
DATP loading was the shift of the amide band at 1639 to
1650−1666 cm−1. The highly negative zeta potential of DATPa−c
compared to their CS−DATPa−c nanocomposites insures the ability of
capturing positively charged chitosan nanoparticles via ionic interac-
tions (Table 1).
It was reported that XRD spectra of pure chitosan have two pro-
minent crystalline peaks at (2θ) of 10° and 20° assigned for the presence
of plenty of OH and NH2 groups that forms strong inter and in-
tramolecular hydrogen bonds [31]. The XRD peak depends on the
crystal size; but in the present study, for all the drug-loaded con-
centrations, the characteristic peak of thienopyridine could overlap
with the polymer itself. Cross-linking Chitosan by TPP (Fig. 3) reveals
the disappearance of peak at 10° and a shift of peak at 20° to 18° and a
new broader one at 25° were observed. This could be attributed to the
rearrangement of molecules in the crystal lattice. It was reported that
after ionic cross-linking CS with TPP, no peak is detected in the dif-
fractograms of CS nanoparticles, reflecting the destruction of the CS
packing structure [32]. In the case of CS−DATPa−c nanocomposites,
several diffraction sharp peaks are observed at 10.4°, 12°, 16°, 21°, 22°
and 25°due to the crystalline phase of the thienopyridine. Compared to
CS−DATPa, the sharp peaks for DATPa were observed at the same
positions indicating the inclusion of DATPa in CS nanoparticles. Also,
the peak for CS nanoparticles at 16° was shifted to 25°. Similar results
were observed for the other thienopyridines (Fig. 3). This confirms the
adsorption of the thienopyridines on the surface of CS nanoparticles.
The peaks of the thienopyridines have disappeared after loading in the
3.3. In vitro drug release
We investigate the potential applications of CS−DATPa−c nano-
composite conjugates as pharmaceutically active compounds to
Fig. 5. Percentage cumulative release of different loading drugs as a function of time; at different pH media; a) pH = 1.2, b) pH = 7.4 and c) pH = 9.
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H. Mansour, et al.
CarbohydrateResearch492(2020)107990
Fig. 6. Representation of the fractional released for all CS−DATPa−c nanocomposites.
Fig. 7. Representation of release exponent (n) and kinetic constants (K) or drug release from the corresponding nanocomposites at 37 °C.
overcome the fast dissolution of chitosan in the stomach and also the
Table 2
Kinetic Constants (K) and release exponents (n) for drug release from the cor-
limited capacity for controlling the release of drugs [34,35]. The in vitro
hydrolysis behavior of CS−DATPa−c nanocomposite was followed by
monitoring the DATP using UV spectrophotometry at 378 nm during
dissolution and diffusion in aqueous solution at different pH media
[36,37]. The cumulative release rates of CS−DATPa−c nanocomposites
as a function of time (t) at different pH media are depicted in Fig. 5.
Fig. 5 showed a first fast release step of DATP molecules and the release
was trapped off with time. This is due to the increased difficulty of
diffusion of the drug included inside the matrix with the decrease of the
initial drug concentration in the crosslinked nanoparticles [38]. The
release of DATP continues for 35 h, showing about 50%–70% of all
CS−DATPa−c nanocomposite. The release profile can be discussed as a
rapid dissolution of DATPa−c followed by release from the
responding CS−DATPa−c nanocomposites at 37 °C.
pH
n
k
Compound
CS−DATPa
1.2
7.4
9
0.41
0.34
0.16
0.37
0.20
0.18
0.48
0.29
0.20
9
25
55
8
CS−DATPb
CS−DATPc
1.2
7.4
9
37
54
8
1.2
7.4
9
28
45
7

H. Mansour, et al.
CarbohydrateResearch492(2020)107990
Fig. 8. Percentage cumulative release of CS−DATPb nanocomposites in different concentrations a) 0.75 mg/L; b) 0.25 mg/L as a function of time and pH.
Table 3
Inhibition zone values of DATPa-c and CS−DATPa−c nanocomposites against
gram-negative (E. coli and P. aeruginosa) and gram-positive (S. aureus and E.
faecalis) bacteria (μg/ml).
E. Coli
P. aeuroguinsa
S. aurins
E. Faecalis
DATPa
0
0
6
0
DATPb
5
0
5
4
DATPc
6
1
5
5
CS−DATPa
CS−DATPb
CS−DATPc
15
16
18
11
8
20
8
8
10
10
10
10
nanocomposites [39]. Effect of pH media on the drugs release behaviors
were investigated and the drug release rate trend in different pH media
is as follow: basic
> neutral > acidic (Figs. 6 and 7). In acidic
medium, most of the amino groups in the nanocomposites were pro-
tonated and the hydrogen bonds between matrix and DATP will be
more favorable. The fast release rate in basic medium, compared to
acidic and neutral, prolongs drug delivery in gastric pH. This would
decrease physiological toxicity resulting from the fast release of the
drug and may find biomedical applications. To investigate the release
mechanism, the data were analyzed using Korsmeyer-Peppas Equation
(2) [40,41].
Fig. 9. Inhibition zone values against gram negative (E. coli and P. aeruginosa)
and gram positive (S. aureus and E. faecalis) bacteria (μg/ml).
(weight) available for release, t is the release time, k is a constant re-
lated to the properties of the drug delivery system, and n is the release
exponent. It was reported that one of the following four mechanisms are
valid for drug release and this depends on the exponent n value [41]; i)
F = Mt/M∞ = ktn → lnF = lnK – n lnt
(2)
where the release fraction (F) is defined as Mt/M∞, Mt is the amount of
DATP drug released at any given time, M∞ is the maximum amount
diffusion through the matrix by
a Fickian diffusion mechanism
(n < 0.5); ii) non-Fickian mechanism (0.5 < n < 1); iii) a zero-order
8

H. Mansour, et al.
CarbohydrateResearch492(2020)107990
Fig. 10. The surviving cell number values versus concentrations of nanocomposites against gram-negative (E. coli and P. aeruginosa) and gram-positive (S. aureus and
E. faecalis) bacteria (μg/ml).
mechanism (n = 1) and non-Fickian super release mechanism (n > 1).
In our case, the n values, determined from Eq. (2), shown in Table 2,
is < 0.5. This suggests a Fickian diffusion mechanism for all drugs
release from the nanoparticles.
inhibition zone varied according to the active group in the copolymer
and also the examined microorganism (Table 3). All bacteria were ex-
posed to different concentrations of the tested compounds with dura-
tion of 24 h and the results are shown in Figs. 9 and 10. The results
showed that: i) the effects of compounds depend on the concentration
and the survival of bacteria increases with increased concentration of
compounds (Fig. 10); ii) the diameter of inhibition zone and the MIC,
the minimal inhibitory concentration, the lowest concentration of the
antimicrobial that will inhibit the visible growth of microorganisms,
varied slightly according to the active group of both DATPa−c and
CS−DATPa−c nanocomposites and also the examined microorganism;
iii) CS−DATPa−c nanocomposites exhibited better activity against
This behavior suggests that the release process is controlled by
diffusion only [42] as the solvent mobility is very low in comparison to
the relaxation rate. The release results will ensure the maximum
availability of the drugs in the colon.
Fig. 8 showed that the drug release is dependent on the con-
centration of nanocomposites. Drug release behavior is prevented by
inter-ionic interaction, which is related to nano chitosan concentration.
As the nano chitosan concentration increases, the crosslinking density
increases due to electrostatic attraction of a larger amount of nano
chitosan with tripolysodium phosphate as a crosslinker agent, and as a
result, physical entanglement among chitosan chains leads to the
compact core and compact polyelectrolyte complex film. These factors
would be expected to slow drug release–erosion process [43]. There-
fore, the slower release rate for CS−DATPb nanocomposites obtained
at higher chitosan concentration can be easily understood.
both Gram-positive and Gram−negative bacteria compared to DATPa-c
.
The higher antibacterial activity of CS-DATPa-c nanocomposites com-
pared to DATPa-c may be explained as follow: i) positively charged
+
CS−DATPa−c nanocomposites (–NH2 on the C−2 position of the
D−glucosamine) can bind to bacterial cell surface which is negatively
charged and disrupt the normal functions of the membrane, e.g. by
promoting the leakage of intracellular components or by inhibiting the
transport of nutrients into cells [44]; ii) or being adsorbed to the cell
membrane through electrostatic interactions, probably by hydrophobic
bonds or hydrogen bridges, being this adsorption con-
centration−dependent causing precipitation and coagulation of cyto-
plasmic proteins and bacterial death [45–48]; iii) also, the association
of thienopyridine in the CS−DATPa−c nanocomposites may affect the
size of the nanocomposite and improves the cellular uptake of the
3.4. Antibacterial activity
In the present study, the antibacterial property of the CS−DATPa−c
nanocomposites was evaluated against Gram−negative (E. coli, P.
aeuroguinsa) and Gram-positive bacteria (S. aureus, E. faecalis) and
compared with DATPa−c. It was found that the diameter of the
9

H. Mansour, et al.
CarbohydrateResearch492(2020)107990
thienopyridine because the antibacterial activity strongly depends on
the particle size [6,48]. The results also indicated that Gram-positive
bacteria were less sensitive to the thienopyridine-chitosan nano-
composites than Gram−negative bacteria. An explanation may be the
different characteristics of the cell surfaces of bacteria. Gram-negative
bacteria have a higher negative charge on the cell surface compared to
Gram−positive bacteria. As a result of the higher negative charge on
the cell surface, the interaction between Gram−negative bacteria and
CS−DATPa−c nanocomposites was stronger than that of Gram−posi-
tive bacteria [49,50].
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