Co-delivery of imidazolium Zn(II)salen and Origanum Syriacum essential oil by shrimp chitosan nanoparticles for antimicrobial applications

Article abstract of DOI:10.1016/j.carbpol.2021.117834

This study reports preparation and physicochemical characterization of natural antimicrobials (Origanum Syriacum essential oil (OSEO), shrimp chitosan nanoparticles (CSNPs)) and new imidazolium ionic liquid-supported Zn(II)Salen. These antimicrobials were

Full text of DOI:10.1016/j.carbpol.2021.117834

Carbohydrate Polymers 260 (2021) 117834
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Co-delivery of imidazolium Zn(II)salen and Origanum Syriacum essential oil
by shrimp chitosan nanoparticles for antimicrobial applications
a,
b
c
d
e,
f,
g
Yasser A. Hassan , Amgad I.M. Khedr , J. Alkabli , Reda F.M. Elshaarawy *, Ali M. Nasr
a
Department of Pharmaceutics, Faculty of Pharmacy, Delta University for Science and Technology, Gamasa, Egypt
b
Department of Pharmaceutics, College of Pharmacy, Al-Bayan University, Baghdad, Iraq
c
Department of Pharmacognosy, Faculty of Pharmacy, Port Said University, 42526, Port Said, Egypt
d
Department of Chemistry, College of Sciences and Arts – Alkamil, University of Jeddah, Jeddah, 23218, Saudi Arabia
Department of Chemistry, Faculty of Science, Suez University, 43533, Suez, Egypt
Institut für Anorganische Chemie und Strukturchemie, Heinrich-Heine Universit a¨ t Düsseldorf, Düsseldorf, Germany
e
f
g
Department of Pharmaceutics, Faculty of Pharmacy, Port Said University, 42526, Port Said, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
This study reports preparation and physicochemical characterization of natural antimicrobials (Origanum
Syriacum essential oil (OSEO), shrimp chitosan nanoparticles (CSNPs)) and new imidazolium ionic liquid-
supported Zn(II)Salen. These antimicrobials were separately or co-encapsulated by CSNPs to fabricate novel
antimicrobial nanoplatforms "NPFs" (OSEO-loaded CSNPs (NPF-1), Zn(II)Salen-loaded CSNPs (NPF-2), and Zn(II)
Salen@OSEO-loaded CSNPs (NPF-3)). The finding of loading, encapsulation, and antimicrobial release studies
confirm the suitability of CSNPs for nanoencapsulation of Zn(II)Salen and OSEO. All NPFs can significantly
suppress the growth of microbial species with performances dependent upon the microbial strain and nano-
platform concentration. The susceptibility of microbes toward new antimicrobials was as follows; Gram-positive
bacteria > Gram-negative bacteria > fungi. The amazing physicochemical features of new nanoplatforms and
their bioactive ingredients (Zn(II)Salen, OSEO, and CSNPs) signify the importance of our designs for developing a
new generation of nanopharmaceuticals supported both natural products and biogenic ionic metal cofactors,
targeting the multidrug resistant (MDR) pathogens.
Essential oil
Imidazolium Zn(II)Salen
Shrimp chitosan nanoparticles
Hybrid antimicrobial cocktails
1
. Introduction
The dramatic spread of multidrug resistant (MDR) pathogens act as a
Shibamoto, 2012). The pharmacological studies confirmed the
multi-biological functions of EOs where they have been recognized as
antimicrobial, antiviral, antiseptic, antirheumatic, anti-inflammatory,
anticancer, antioxidant, antispasmodic, anti-allergic, anticancer, and
aphrodisiac agents (Ali et al., 2015; Asif, Saleem, & Saadullah, 2020;
Bhardwaj et al., 2020). Therefore, EOs are always beneficial sources of
promising and safe pharmacological candidates, particularly, antimi-
crobials. To that end, numerous studies have been carried out on the
antimicrobial performances of EOs against wide-range of microbial
strains, including food-borne pathogens (FBPs) (Calo, Crandall,
O’Bryan, & Ricke, 2015; Reyes-Jurado et al., 2020; Seow, Yeo, Chung, &
Yuk, 2014).
great challenge for the therapeutic field and a serious global health
threat demanding exploration of novel innovative antimicrobials; able
to induce multiple biological effects into the microbial cells targeting
disabling their defense strategies (Belete, 2019; Ruddaraju, Pammi,
Guntuku, Padavala, & Kolapalli, 2020). Several strategies have been
reported to suppress or totally block these bacterial resistances such as
antimicrobial adjuvants, Nano-encapsulation, therapy with bacterio-
phage particles, and others (Baptista et al., 2018; Darvish, Bahrami,
Jafari, & Williams, 2020; Zaheer et al., 2017).
Across the ages, essential oils (EOs) have attracted great attention as
sources of natural pharmacological products that have considerable
potentials to utilize as alternative safe medications for treating many
infectious and foodborne diseases (Edris, 2007; Shaaban, El-Ghorab, &
Zinc (Zn(II)) is essential for human health where it is a substantial
component of about 10 % of all human proteins and performs catalytic,
structural, and regulatory cellular functions (Krę z˙ ela & Maret, 2016;
Roohani, Hurrell, Kelishadi,
& Schulin, 2013). Moreover, the
*
Corresponding author at: Department of Chemistry, Faculty of Science, Suez University, 43533, Suez, Egypt.
E-mail addresses: reda.elshaarawy@suezuniv.edu.eg, reel@hhu.de (R.F.M. Elshaarawy).
https://doi.org/10.1016/j.carbpol.2021.117834
Received 10 December 2020; Received in revised form 28 January 2021; Accepted 14 February 2021
Available online 18 February 2021
0
144-8617/© 2021 Elsevier Ltd. All rights reserved.

Y.A. Hassan et al.
Carbohydrate Polymers 260 (2021) 117834
antimicrobial efficacy of Zn(II) as an antibacterial (Abendrot et al.,
2.1. Essential oil extraction and analysis
2
020; Atmaca, Gül, & Çiçek, 1998; Joseyphus & Nair, 2008), as well as
an antifungal (Jaafar et al., 2020; Joseyphus & Nair, 2008), were known
for a long time.
2.1.1. Plant material and extraction of O. syriacum essential oil (OSEO)
Samples of Origanum syriacum L. (O. syriacum) plants have been
collected from cultivated plants grown in El Arish, Egypt. The collected
plant materials were air-dried in shade for one week and then manually
cut into small pieces followed by ground into a moderately coarse
powder.
Metallosalens are quite versatile coordination compounds having
many applications such as catalysis, chemical sensing, medicinal, and
pharmacological (Erxleben, 2018; Pessoa & Correia, 2019). Further-
more, previous studies revealed the diverse pharmacological effects for
metallosalens such as; antimicrobial (Musa, Khan, Aspedon, & Cooper-
wood, 2010; Nworie, 2016; Pessoa & Correia, 2019), anticancer
The Egyptian Pharmacopoeia protocol was followed to extract OSEO
from the fresh and pre-dried oregano plant. In brief, 100 g of fresh or
pre-dried powdered Egyptian O. syriacum plant materials were hydro-
(
Abdalla et al., 2020; Erxleben, 2018; Pessoa & Correia, 2019), and
◦
antiviral (Tarasconi, Capacchi, & Pelosi, 2000). Several Zn(II)Salen
distilled at 100 C for 3 h using conventional Clevenger-type hydro-
complexes were reported to have remarkable antimicrobial efficacies
distillation apparatus. The extracted oil was collected, dissolved in
◦
(
Inba, Annaraj, Thalamuthu, & Neelakantan, 2013; Khan, Nami, Bhat,
Kareem, & Nishat, 2017; Mukhopadhyay, Kundu, Porwal, & Mobin,
020). In addition, the biocompatible nature of Zn(II) ions and its salen
hexane, dried over anhydrous Na
use.
2 4
SO , and stored at 4ꢀ 6 C for further
2
complexes coupled with their minimal cytotoxicity make them prom-
ising templates for the development of new pharmacological agents
2.1.2. Analysis of OSEO by gas chromatography-mass spectrometry
analysis (GC–MS)
(
Kumar, Roopa, Bhalla, & Kumar, 2021).
The chemical composition of extracted essential oil was investigated
by GC–MS technique using an HP-5890 II gas chromatograph equipped
with an HP-5972 mass-selective detector in electron impact mode (70
Interestingly, recently it was reported the potential for EOs to act
synergistically with antimicrobials and antioxidants compounds for
significantly enhanced bioactivity (Hadidi, Pouramin, Adinepour
Haghani, & Jafari, 2020; Halevas et al., 2017). Therefore, combinations
of synthetic and natural antimicrobials, such as EOs, may offer one of the
most promising approaches to overcome microbial resistance to anti-
biotics. Nano-encapsulation of bioactive compounds using
biopolymers-based nanomaterials also provide an additional promising
weapon in fighting multidrug resistant infections (Detsi et al., 2020;
Gupta, Mumtaz, Li, Hussain, & Rotello, 2019; Yahya et al., 2020).
Nano-encapsulation can also address drawbacks related to drug solubi-
lity (Detsi et al., 2020).
eV). Analytical conditions: injector and MS transfer line temperatures,
◦
220 and 240 C, respectively; injection, 2
μ
L (10 % hexane solution);
◦
◦
ꢀ 1
oven temperature, 60ꢀ 240 C with heating rate 3 C min ; carrier gas,
ꢀ
1
He at 1 mL min . The constituents of OSEO were identified by com-
parison of their respective relative retention times and mass spectra with
those of authentic samples, on the basis of their relative retention indices
(RRI) to a series of n-alkanes. Further identifications were performed
with aid of the NBS75 K library of mass spectra created from pure
components of known oils, and their respective MS literature data
(Adams, 2001).
The incredible features of chitosan (CS) and thus chitosan nano-
particles (CSNPs) (Divya & Jisha, 2018) make it the best choice in drug
delivery systems (Elbehairi, Ismail, Alfaifi, Elshaarawy, & Hafez, 2020).
To this end, CSNPs are acting as a promising class of bioactive encap-
sulators for many drugs delivers (Divya & Jisha, 2018).
2.2. Synthesis of Salen ligand and its Zn(II) complex
2.2.1. Synthesis of Salen ligand (4)
This ligand was prepared in accordance with our earlier work
(Alfaifi, Elbehairi, Hafez, & Elshaarawy, 2019) with slight modification,
in brief, a solution of isopropylsalicylaldehyde-butylimidazolium chlo-
Inspired by the aforesaid outstanding facts and in the continuity of
our relentless efforts in designing new smart chitosan-based bioactive
materials (Elshaarawy, Refaee, & El-Sawi, 2016, Elshaarawy et al.,
i
n
+
ride (H( Pr)sal( BuIm Cl–), 3) (673.7 mg, 2.0 mmol) in dry ethanol (25
mL) was mixed with an ethanolic solution (10 mL) of ethylenediamine
(60.1 mg, 1.0 mmol) in a Schlenk system under vigorous stirring at room
temperature for 5 h. After completion of the reaction as judged by
thin-layer chromatography (TLC), the solvent was removed in vacuo and
the residue was subjected to a silica gel column chromatography using a
mixture of n-hexane-ethyl acetate (9:1) as an eluent to give a pure salen
ligand (1297.8 mg, 93 % yield) as a pale orange solid which dried and
2
017; Sofy, Hmed, Abd El Haliem, Zein, & Elshaarawy, 2019), the
present study aimed to investigate the synergistic behavior of EO with
new bioactive Zn(II)Salen complex through co-encapsulation by CSNPs,
targeting multifunctional pharmaceutical nanocomposites. Despite
several studies have reported the antimicrobial action of essential oils
enveloped with chitosan matrices (Detsi et al., 2020; Yahya et al., 2020),
nevertheless, no work has been reported for the fabrication of nano-
pharmaceuticals incorporating several pharmacophores (OSEO, metal-
losalen, and imidazolium ionic liquid terminals) which may induce
several biocidal mechanisms inside the microbial cells to overcome the
self-microbial resistance.
◦
preserved in a desiccator for further use, mp 89–91 C. FT-IR (KBr,
ꢀ 1
cm ): 3432 (m, br), 3131 (m, sh), 3069 (m, sh), 2960 (s, sh), 2933 (s,
sh), 2864 (s, sh), 1630 (vs, sh), 1560 (s, sh), 1464 (s, sh), 1382 (m, sh),
1272 (s, sh), 1156 (s, sh), 1099 (s, sh), 953 (w, sh), 862 (m, sh), 773 (m,
sh), 645 (m, sh), 502 (w, br). 1H NMR (200 MHz, DMSO-d
14.02 (s, 2 H), 9.47 (s, 2 H), 8.54 (s, 2 H), 7.83 (d, J =1.97 Hz, 2 H), 7.37
) δ (ppm):
6
2
. Materials and methods
(
d, J =1.58 Hz, 2 H), 7.17 (s, 2 H), 6.98 (s, 2 H), 5.37 (s, 4 H), 4.18 (t, J
Specifications of materials used in this work coupled with their
=7.0 Hz, 4 H), 3.96 (t, J =7.1 Hz, 4 H), 3.44ꢀ 3.33 (m, 2 H), 2.15ꢀ 1.92
(m, 4 H), 1.89ꢀ 1.60 (m, 4 H) 1.16 (d, J =5.2 Hz, 12 H), 0.89 (t, J =7.4
suppliers, different techniques utilized for the comprehensive charac-
terization of the prepared samples (such as Fourier-transform infrared
Hz, 6 H). 13C NMR (125 MHz, DMSO-d ) δ (ppm): 159.21, 157.92,
6
(
(
FTIR), Ultraviolet–Visible (UV–vis), Nuclear magnetic resonance
NMR) spectroscopy, Electrospray ionization mass spectrometry (ESI-
139.64, 138.61, 132.12, 129.75, 127.34, 125.16, 123.96, 122.71, 63.11,
57.05, 48.57, 34.85, 28.33, 23.87, 21.47, 14.42. ESI MS: m/z 662.2
+
+
MS), Thermogravimetric analysis (TGA), and Scanning electron micro-
scopy (SEM)) are shown in the electronic supplementary information
(<5%, [C38
H
ClN O ]
=
[M
+
–
Cl–] ) and 331.1 (20 %,
54
6 2
2
+
[C38H54CN O ] = [M – 2 Cl–] ). Anal. Calcd. for C38H54Cl N O (M =
6
2
2 6 2
(
ESIy). Moreover, the practical protocol used in the preparation of the
new ionic liquid, isopropylsalicylaldehyde-butylmidazolium chloride
3), was also described in the ESIy.
697.79): C, 65.41; H, 7.80; N, 12.04; Found: C, 65.28; H, 7.83; N, 12.07.
(
2.2.2. Synthesis of Zn(II)Salen complex
A solution of the salen-bis(imidazolium) salts H
Cl–) (4) was prepared by dissolving a certain amount of salen ligand
0.7 g, 1 mmol) in ethanol (10 mL). Then a solution of ZnCl (0.14 g, 1
(iPr)
salen(BuIm -
+
2 2
2
(
2
2

Y.A. Hassan et al.
Carbohydrate Polymers 260 (2021) 117834
◦
mmol) in ethanol (5 mL) was added to the previous solution with stirring
and the reaction mixture was further stirred under reflux for 1 h.
Thereafter, the solvent was partially evaporated under reduced pressure
and the residue was kept at room temperature to crystallize. The ob-
Salen@CSNPs (NPF-2) which stored at 4 C for further analysis.
2.3.3. Synthesis of [Zn(II)Salen]@OSEO-loaded CSNPs (NPF-3)
The [Zn(II)Salen]@OSEO-loaded CSNPs was synthesized following
the aforesaid protocol for OSEO@CSNPs and Zn(II)Salen@CSNPs
nanocomposites. In brief, a solution of OSEO (0.5 mL) in DCM (4 mL)
was gradually added to the CS gel (vide supra) under vigorous stirring at
RT. Subsequently, an aqueous solution of Zn(II)Salen complex (0.10 g)
i
+
tained product, [Zn(II)( Pr)
2
salen(BuIm -Cl–)
2 2
].H O, was collected by
filtration, washed with cooled absolute ethanol (3 × 3 mL), recrystal-
lized from acetonitrile and dried under vacuum. Zn(II)salen complex is
ꢀ 1
obtained as pale yellow powder (0.6 g, 78 %). FT-IR (KBr, cm ): 3440
s, br), 3133 (m, sh), 3098 (m, sh), 2955 (s, sh), 2935 (s, sh), 2865 (s, sh),
617 (vs, sh), 1577 (s, sh), 1438 (s, sh), 1388 (m, sh), 1283 (s, sh), 1155
s, sh), 1103 (s, sh), 957 (w, sh), 832 (m, sh), 780 (m, sh), 667 (m, sh),
(
in Milli-Q H
2
O (8 mL, 1:1 v/v) was gradually added at the same con-
1
ditions. Thereafter, follow the aforesaid protocol.
(
6
23 (m, sh), 570 (w, br), 470 (w, br), 444 (w, br). ESI-MS: m/z 743.4
2
.4. Determination of encapsulation efficiency and loading capacity
+
+
(
<5%, [C38
H
54ClN
6
O
3
Zn]
=
[M
–
Cl–] ) and 354.1 (23 %,
Zn]2 = [M – 2 Cl–] ). Anal. Calcd. for C38
+
+
[
C
38
H54CN
6
O
3
2 6 3
H54Cl N O Zn
All experiments were carried out in triplicates and the results were
(
M = 779.17): C, 58.58; H, 6.99; N, 10.79; Found: C, 58.48; H, 7.03; N,
expressed as mean ± standard deviation (SD).
1
2
2
0.73.
2
.4.1. [Zn(II)Salen] encapsulation efficiency and loading capacity
.3. Synthesis of CSNP-based nanoplatforms (NPFs)
.3.1. Synthesis of OSEO-loaded CSNPs (NPF-1)
The flame atomic absorption spectrophotometry (FAAS) was used to
estimate the percentage of [Zn(II)Salen] complex encapsulated by
CSNPs in each nanoplatforms (NPF-2 and NPF-3). A 0.25 g dry sample
was dissolved in 20 mL of 6 N HCl in a 100 mL PTFE beaker and the
mixture was boiled until complete dissolution of the sample (1 h).
Thereafter, the solution was cooled to RT and transferred to a 200 mL
volumetric flask for appropriate dilutions, to determine the Zn(II) con-
tent according to the standard method 3111 B (Clesceri, Greenberg, &
Trussell, 1989). The encapsulation efficiency of [Zn(II)Salen] (EE%) was
calculated based on the following equation (Eq. (1)):
0
2
.20 g of shrimp CS powder was dissolved in 20 mL of Milli-Q H O
containing 0.20 mL of glacial acetic at room temperature (RT) under
◦
vigorous stirring. The resulting solution was further stirred at 40 C for
2
4 h to obtain a colorless gel. This gel was cooled to ambient tempera-
ture and then centrifuged at 8500 rpm for 30 min, to remove any
insoluble material. Thereafter, the clear gelatinous solution was re-
◦
heated to 50 C followed by the addition of 0.25 mL of the surfactant
(
Tween 20) under continuous stirring, and the resulting mixture was
m
m
e
t
further stirred at the same temperature for 2 h to obtain a homogeneous
mixture. Afterward, OSEO (0.5 mL) was dissolved separately in
dichloromethane (DCM) (4 mL) by stirring at RT to obtain a clear so-
lution. The resulting oil solution was gradually dropped into CS solution
under vigorous stirring at ambient temperature. Complete homogeni-
zation was achieved by stirring the reaction mixture in an ice bath for 50
min to get, initially, oil-in-water (O/W) emulsion which further stirred
for an additional 1 h at the same conditions for complete homogeniza-
tion. Following homogenization, an aqueous TPP solution (0.2 % (w/v),
EE% =
x 100
(1)
Where m
e
and m
t
are the amounts of encapsulated and initially added
[
Zn(II)Salen] complex, respectively.
The loading efficiency of [Zn(II)Salen] into CSNPs (LC%) was
calculated using the following equation (Eq. (2)):
m
W
e
LC% =
x 100
NPs
(2)
2
0 mL) was added dropwise to the reaction vessel under vigorous stir-
Where m
e
is the amount of encapsulated [Zn(II)Salen] complex and
ring and then the whole mixture was further stirred for an additional 1 h
W
NPs is the weight of CSNPs after freeze drying.
in an ice bath. The resulting white nanocomposite (OSEO@CSNPs, NPF-
◦
1
) was collected by centrifugation at 8500 rpm for 30 min at 4 C and
2
.4.2. OSEO encapsulation efficiency and loading capacity
washed with Milli-Q water (3 × 5 mL). Then, the centrifuged nano-
composite was re-dispersed in 20 mL of Milli-Q H O via ultrasonication
The percentage of OSEO encapsulated by CSNPs was estimated using
2
UV–vis spectrophotometry at the wavelength characteristic for OSEO
(260 nm) (Fig. S1, ESIy). A certain amount (10 mg) of OSEO@CSNPs or
[Zn(II)Salen]@OSEO@CSNPs nanoplatforms (NPF-1 and NPF-3) was
in an ice bath for 10 min to obtain a homogeneous suspension. Even-
tually, the suspension was freeze-dried at ꢀ 35 ◦C for 72 h to produce
◦
OSEO-loaded CSNPs (NPF-1) which stored at ꢀ 20 C for further anal-
3 3
ground and immersed into a mixture of HNO -EtOH (HNO :EtOH, 2:1 v/
ysis, to avoid their agglomeration (Hasheminejad, Khodaiyan, & Safari,
v, 6 mL). Then, the reaction mixture was vigorously vortexed for 10 min
followed by centrifugation at 8500 rpm for 10 min to allow settling
down of liberated CSNPs after releasing of OSEO or [Zn(II)Salen]
2
019). Nascent CSNPs, without OSEO, were prepared by the same pro-
tocol to the optimized CSNP formulation.
ꢀ 1
@
OSEO into the solution. The content of OSEO (mg mL ) in each
2
.3.2. Synthesis of [Zn(II)Salen]-loaded CSNPs (NPF-2)
0 mL of an aqueous solution of TPP (0.2 % w/v) was added drop-
nanocomposite was estimated by measuring the absorbance values at
2
2
60 nm for the isolated supernatant from each experiment.
wise to the resulting CS gel (vide supra) under vigorous stirring and the
mixture was stirred for 1 h in an ice bath. Subsequently, a solution of [Zn
The encapsulation efficiency of OSEO (EE%) was calculated based on
e t
the following equation (Eq. (1)), where, in this case, m and m are the
(
II)Salen] complex (0.10 g) in a mixture of DCM and Milli-Q H
2
O (8 mL,
concentrations of encapsulated and initially added essential oil,
respectively. On the other hand, The loading capacity of OSEO into
CSNPs (LC%) was calculated using the following equation (Eq. (2)),
1
:1 v/v) was gradually added to the gelatinous CS solution under
vigorous stirring at RT. Thereafter, the reaction mixture was stirred in an
ice bath for 50 min to obtain oil-in-water (O/W) emulsion which further
stirred for an additional 1 h at the same conditions to achieve complete
e
where m is the amount of encapsulated OSEO and WNPs is the weight of
CSNPs after freeze-drying. All experiments were carried out in triplicates
homogenization.
Subsequently,
the
nanocomposite
(Zn(II)
and the results were expressed as mean ± standard deviation (SD).
Salen@CSNPs, NPF-2) was formed as a yellow fine product that was
◦
gathered by centrifugation at 8500 rpm for 30 min at 4 C and washed
with Milli-Q water (3 × 5 mL). Afterward, the collected nanocomposite
2.5. In vitro release study
2
was re-dispersed in Milli-Q H O (20 mL) under ultrasonic irradiation in
an ice bath for 10 min to obtain a homogeneous suspension. Eventually,
All experiments were carried out in triplicates and the results were
this suspension was freeze-dried at ꢀ 35 ◦C for 72 h to yield Zn(II)
expressed as mean ± standard deviation (SD).
3

Y.A. Hassan et al.
Carbohydrate Polymers 260 (2021) 117834
2
.5.1. [Zn(II)Salen] release study
{Escherichia coli (EC, RCMB 000103), Pseudomonas aeruginosa (PA,
RCMB 000102)}; and fungal strains {Aspergillus flavus (AF, RCMB
02782), Candida albicans (CA, RCMB 05036)}.
A certain amount (20 mg) of [Zn(II)Salen]-based nanoplatforms
(
NPF-2 and NPF-3) was ground and immersed into 5 mL of release
medium {oxygenated saline solution of composition (mM): 136.0 NaCl,
KCl, 2.4 CaCl , 1.1 MgCl , 1.0 NaHCO , 11.0 glucose, 10.0 4-(2-
7
2
2
3
2
.7. Statistical analysis
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 40 %
EtOH}(Halevas et al., 2017) under continuous stirring (300 rpm) at 37 ±
◦
The SPSS statistics software (version 20.0) (SPSS Inc.) was used to
1
C. Aliquots of 2 mL were withdrawn with a syringe at fixed time in-
perform all statistical assays. The collected results were analyzed using
one-way variance (ANOVA) followed by a multi-range test. Data figures
have represented using the Origin 9.1 program (SR0 b215). All data
were expressed as mean ± standard error, and the significance level was
considered for P-value < 0.05.
tervals for Zn(II) spectrophotometric analysis. After removing the
insoluble nanocomposite by centrifugation (13,000 rpm, 1 min), the
clear solution was analyzed by FAAS at 213.9 nm. The percentage of Zn
(
II)Salen complex released (R%) was calculated based on the following
equation (Eq. (3)) (Li, Zhao, Lv, & Liu, 2007);
R% = C
i
ꢀ C
x 100
(3)
3. Results and discussion
C
i
3
.1. Synthesis chemistry
where C
i
and C represent initial and liberated Zn(II) concentrations in
the control and releasing medium solutions, respectively.
The bis(imidazolium)-salen ligand has synthesized starting from 2-
isopropylphenol following our previous designed protocol (Elshaar-
awy, Kheiralla, Rushdy, & Janiak, 2014) with slight modification (see
Scheme 1). Initially, 3-iso-propylsalicylaldehyde (1) was obtained by the
ortho-formylation reaction of 2-iso-propylphenol under ultra-dry con-
ditions using paraformaldehyde in presence of catalytic amounts of
magnesium dichloride and triethylamine. Thereafter, 3-iso-propylsalicy-
laldehyde was chloromethylated using a mixture of paraformaldehy-
2
.5.2. OSEO release study
The releasing profile of OSEO from nanoplatforms (NPF-1 and NPF-
3
) was investigated as aforesaid in Section 2.5.1. After removing insol-
uble nanocomposite by centrifugation (13,000 rpm, 1 min), the clear
solution was analyzed for OSEO content using UV–vis spectroscopic
measurements at 260 nm.
2
de-ZnCl -HCl to yield the corresponding chloromethylated derivative
i
2
.6. Antimicrobial assessment
(2). Subsequently, the new salicyl-imidazolium ionic liquid (( Pr)sal
n
+
(
BuIm -Cl–), 3) was prepared by the quarternization process of
n
The antimicrobial activity of OSEO, [Zn(II)Salen], and their encap-
1- butylimidazole using the chloromethylated iso-propylsalicylaldehyde
i
sulated forms by CSNPs was assessed according to our earlier modified
well diffusion susceptibility test (Elshaarawy & Janiak, 2016). The
well-diffusion assay was performed on Muller-Hinton agar (MHA) plates
against Gram-positive bacteria {Staphylococcus aureus (SA, RCMB
(2). Eventually, a Schiff base condensation reaction between ( Pr)sal
n
+
( BuIm -Cl–) (3) and ethylenediamine was applied to obtain the desired
i
n
+
salen ligand (H
2
( Pr)
2
salen( Bu
2
Im -Cl–)
2
, 4). This salen ligand was
used as a chelating ligand for Zn(II) ion to prepare the corresponding Zn
i
n
+
0
00106), Bacillus subtilis (BS, RCMB 000101)}; Gram-negative bacteria
(II)salen complex, [Zn(II){( Pr)
2
salen( Bu
2
Im -Cl–)
2
}]. The salen-bis
i
n
+
Scheme 1. Schematic diagram for extraction of O. syriacum essential oil (OSEO) and Synthesis of: salicyl-imidazolium ionic liquid (( Pr)sal( BuIm -Cl–), 3), salen-bis
i
n
+
i
n
+
(
imidazolium) chloride ligand (H
2
( Pr)
2
salen( Bu
2
Im -Cl–)
2
, 4), and Zn(II)salen complex [Zn(II){( Pr)
2
salen( Bu
2
Im -Cl–)
2
}].
4

Y.A. Hassan et al.
Carbohydrate Polymers 260 (2021) 117834
(
imidazolium) chloride (4) and its Zn(II) complex were isolated in high
3.2. Chemical composition of OSEO
1
yields and characterized by elemental and spectral (FTIR, UV–vis,
NMR, C NMR, ESI-MS) analysis.
H
1
3
A 5.9 % yield of essential oil (OSEO) was collected from the
O. syriacum plant. The chemical composition of this oil was investigated
using GC–MS analysis. The chemical constituents of OSEO, obtained
from cultivated O. syriacum plant gathered from El Arish, Egypt, have
presented in Table 1. Twenty compounds with an overall percentage of
99.61 have been detected in the OSEO, as revealed from GC–MS anal-
ysis. As shown in Table 1, the composition of OSEO was dominated by a
great ratio of oxygenated monoterpenes (92.14 %) represented in
Carvacrol (61.18 %) and Thymol (29.85 %) as the major components.
Other oxygenated monoterpenes are participated by few amounts in the
formulation of OSEO (see Table 1). On the other hand, the biogenetic
precursors (monoterpene hydrocarbons) exhibited low contribution
(6.98 %) in OSEO composition. For instance, the major compound of
these biogenetic precursors in OSEO is δ-Terpinene, representing only
1.68 % of the total oil. Also, a bicyclic sesquiterpene hydrocarbon
(β-Caryophyllene, 0.11 %), oxygenated sesquiterpene (Caryophyllene
On the other hand, the desired encapsulating-delivering agent
CSNPs) was formulated by the ionotropic gelation process of the
(
cationic CS polyelectrolyte using sodium tripolyphosphate (STPP) as a
crosslinking agent. The gelation process has occurred via electrostatic
interaction between the quaternized amine groups of CS and negatively-
charged counterion (TPP) (Koukaras, Papadimitriou, Bikiaris, & Frou-
dakis, 2012) (Scheme 2). Interestingly that Tween 20 was incorporated
into the matrix of CSNPs during the ionotropic gelation process to
fine-tune their morphology, stability, and hydrophilicity (Halevas et al.,
2
017). Tween 20 can bind to the surface of CSNPs through H-bonding
interactions forming a CSNPs-Tween 20 semi-interpenetrating network
(
Halevas et al., 2017).
Finally, the CSNPs-based nanoplatforms (OSEO@CSNPs, [Zn(II)
Salen]@CSNPs, and [Zn(II)Salen]@OSEO@CSNPs) were assembled by
the nanoencapsulation EO, Zn complex and their combinations could be
assembled into CSNPs-Tween network through; (i) H-bonding between
the functional groups in OSEO or Zn(II)salen and the OH and NH groups
of CSNPs (Li et al., 2017). (ii) Hydrophobic interactions between oil
molecule and the lipophilic side chains of Tween (Li et al., 2017)
oxide, 0.09 %; -Cadinol, 0.11 %), and tetracyclic diterpenoid (Long-
ι
ipenol, 0.18 %) have shared in the formulation of oreganum oil by a low
extent.
(
Scheme 2).
Scheme 2. Schematic illustration for preparation of (A) CSNPs and [Zn(II)Salen]@CSNPs-Tween 20 (NPF-2). (B) Tween 20-suported CS (CS-Tween 20), CSNPs-
based nanoplatforms (OSEO@CSNPs-Tween 20 (NPF-1) and [Zn(II)Salen]@OSEO@CSNPs-Tween 20 (NPF-3).
5

Y.A. Hassan et al.
Carbohydrate Polymers 260 (2021) 117834
Table 1
their successful formation. Where, the demise of carbonyl vibration
–
1
Chemical composition of the essential oil from the cultivated Egyptian
band (1644 cm in the ionic liquid spectrum, Fig. S2) from the spectrum
of the salen ligand coupled with the emergence of a new stretch at 1629
O. Syriacum plant.
–
1
cm (Fig. S3), assignable for the vibration of azomethine group (H C =
–
Compound
RT (min)
Yield (%)
N), confirms the success of preparation of the bis(imidazolium)-salen
ligand (4) (Elshaarawy et al., 2014). On the other hand, the wave-
number values for the vibration of the azomethine and aryl-O groups
Monoterpene hydrocarbons
■
■
■
■
■
■
■
■
■
■
α
-Thujene
- Pinene
4.22
4.67
5.15
5.34
5.77
6.23
7.01
7.40
8.41
9.27
0.06
0.64
0.12
1.04
tr
α
–
1
Camphene
p-Cymene
Sabinene
have decreased to 1619 and 1259 cm , respectively, (Fig. 1-(A)) as
–1
compared to the parent ligand (1629 and 1270 cm , respectively)
indicating their binding to the Zn(II) ion, forming Zn(II)salen complex
β-Myrcene
1.32
1.24
0.37
1.98
0.21
i
n
+
α
-Terpinene
[Zn(II){( Pr)
2
salen( Bu
2
Im -Cl–)
2
}] (Halevas et al., 2017). Furthermore,
–
1
Limonene
the emergence of new vibrational modes at 534 and 437 cm , attrib-
utable to vibrations of Zn–O and Zn–N fragments (Hosseinzadeh,
Khorshidi, & Janczak, 2020), is further support for chelation of Zn(II)
ion by the phenolic oxygen and azomethinic nitrogen of salen ligand. In
δ-Terpinene
Terpinolene
Oxygenated Monoterpenes
■
■
■
■
■
■
1-Octen-3-ol
Linalool
9.41
0.22
0.22
61.18
0.89
29.63
tr
–
1
9.90
addition, the broad band centered at 3440 cm confirms the hydrating
nature of the complex as proposed by the microanalytical results of
complex (see Experimental part). Noteworthy, observation of vibra-
Carvacrol
4-Terpineol
Thymol
12.54
13.01
14.52
16.71
–
1
tional bands around 864 and 754 cm in the spectra of free ligand and
its complex, due to the in-plane and out-plane flexible vibrations of the
imidazolium ring, proves supporting of both ligand and complex with
imidazolium ionic liquid terminals (Elshaarawy et al., 2014).
Carvone
Sesquiterpene hydrocarbons
β-Caryophyllene
Oxygenated Sesquiterpene
■
17.40
0.11
■
■
Caryophyllene oxide
-Cadinol
24.07
26.37
0.09
0.11
The FT-IR spectrum of OSEO (Fig. 1-(B)) shows a broad vibrational
ι
ꢀ 1
band centered at 3459 cm owing to the stretching vibration mode of
alcoholic OH group of oxygenated terpenes (1-Octen-3-ol, Linalool,
Carvacrol, 4-Terpineol, Thymol, and Cadinol). Moreover, sharp peaks
Diterpenes
■
Longipenol
29.99
0.18
Total
99.61
ꢀ 1
were observed at 2963, 2918, and 2872 cm that distinctive for the
stretching vibrations of aliphatic methyl (CH ), methylene (CH ), and
methine (CH) fragments of terpene backbone (Guti ´e rrez-Pacheco et al.,
Tr = Trace, relative percentage less than.0.025 %
3
2
3
3
.3. Structural and morphological characterization of new materials
ꢀ
1
–
2
020). Absorption at 1634 cm can be related to the C
–
O stretching
vibration, probably from carvone present in the OSEO. Shoulders at
.3.1. Elemental analyses
ꢀ
1
(iPr)
n
+
about 3022 and 1601 cm are attributed to the vibration of the olefinic
Bis(imidazolium)-salen ligand (H
2
2
salen( Bu
2
Im -Cl–)
2
, 4) and
–
i
n
+
–
C H and CC
fragments, respectively, of the terpenoid backbone. Also,
OC stretching vibration can be
its Zn(II) complex [Zn(II){( Pr)
2
salen( Bu
2
Im -Cl–)
2
}] gave satisfactory
the absorption bands for the C
noticed at 1261 and 1062 cm (Guti ´e rrez-Pacheco et al., 2020).
As shown in the spectrum of CSNPs (Fig. 1-(C)), main peaks can be
–
1
–
C, H, N elemental analyses which are in full agreement with the sug-
gested structural formula for the ligand and its chelate (see Experimental
part).
ꢀ
ꢀ 1
seen at 3433, 1647, 1595, and 1158 cm which are attributable to the
vibration of O
–
H & NH, amide I (CO––), bending NH
, and β-glyco-
–
2
3
.3.2. FT-IR
sidic (C-O-C) groups, respectively, of CS skeleton (Elshaarawy, Mustafa,
The FT-IR spectra of [Zn(II)Salen], CSNPs, OSEO, OSEO-loaded
Herbst, Farag, & Janiak, 2016). In addition, the characteristic bands at
CSNPs (NPF-1), Zn(II)Salen-loaded CSNPs (NPF-2), and Zn(II)
Salen@OSEO-loaded CSNPs (NPF-3) are given in Fig. 1. The FT-IR
spectra of salicyl-imidazolium ionic liquid (3) and salen-bis
ꢀ 1
1
256, 1158, 1088, and 897 cm owing to the stretching vibrations of
O, PO , PO , and P OP groups of TPP is indicative of the success
P
–
2
3
– –
of TPP-CS crosslinking, forming CSNPs (Elbehairi et al., 2020; Martins,
(
imidazolium) chloride ligand (4) (Figs, S2, S3, ESIy) have evident
de Oliveira, Pereira, Rubira, & Muniz, 2012).
The spectrum of OSEO-loaded CSNPs (Fig. 1-(D)) has revealed
coexisting of IR spectral peaks characteristic for the bare CSNPs, Tween
2
0, and OSEO together, however, with some changes in their positions
and/ or intensities owing to the mutual interactions between these
components. To name few, the characteristic stretches of OSEO (vide
ꢀ 1
supra) were either red-shifted to be seen at 3428, 2936, 2852 cm or
ꢀ 1
blue-shifted to be positioned at 1660, 1269 cm
coupled with
decreasing of their intensities in the spectrum of its encapsulated form.
Meanwhile, a set of peaks distinctive for CSNPs (vide supra) was also
observed in the spectrum of OSEO-loaded CSNPs but with slight fluc-
tuations in their sites and/ or intensities, for instance; the peaks of amide
I, β-glycoside, P
–
O, and PO
3
groups were noticed at 1660, 1126 1269,
ꢀ 1
and 1024 cm , respectively. Additionally, the emergence of a new peak
ꢀ
1
–
–
at 1738 cm (assignable for ester C O) indicates the sharing of Tween
0 in the nanoencapsulation of OSEO by CSNPs (Halevas et al., 2017).
2
The spectrum of Zn(II)salen-loaded CSNPs (Fig. 1-(E)) has denoted
the nanoencapsulation of this complex by CSNPs as revealed from the
wavenumber-shift of azomethine, hydroxyl, and other groups to higher
values in comparison to the nascent Zn(II)salen complex. For example,
the azomethine (H
–
C = N) group of Zn(II) complex was observed at
Fig. 1. FT-IR spectra of (A) Zn(II)salen complex, (B) OSEO, (C) CSNPs, (D)
OSEO@CSNPs (NPF-1), (E) Zn(II)salen@CSNPs (NPF-2), and (F) Zn(II)sale-
n@OSEO@CSNPs (NPF-3).
ꢀ 1
1
641 cm in the spectrum of the encapsulated form (Δ
free = +22 cm ), whereas, the in-plane and out-plane vibrational
ν
=
ν
encapsulated
–
ꢀ 1
ν
6

Y.A. Hassan et al.
Carbohydrate Polymers 260 (2021) 117834
bands of the imidazolium ring can be seen at 868 and 749 cm–1. Also, the
step (164–475 ◦C), the significant weight loss can be attributed to
several reactions such as dehydration of the glucopyranose rings, partial
depolymerization, decomposition of the acetylated and deacetylated
units of the chitosan backbone (Halevas et al., 2017). Also, OSEO-loaded
CSNPs (NPF-1) displayed thermal behavior similar to that of CSNPs as it
peak characteristic for OH & NH and β-glycoside groups of CSNPs can be
ꢀ
1
ꢀ 1
seen at 3445 and 1144 cm (Δ
ν
= +11 and –14 cm ), respectively.
Changes in the intensity of these peaks in comparison to the corre-
sponding absorption in the spectra of bare CSNPs and Zn(II)salen in-
dicates the successful encapsulation of complex by CSNPs.
◦
is also thermally stable up to 50 C and exhibited two-stage degradation
In the same context, the spectrum of Zn(II)salen@OSEO-loaded
CSNPs (Fig. 1-(F)) was dominated by four sets of vibrational peaks
attributable for the functional groups of Zn(II)salen, OSEO, Tween 200,
and CSNPs, however, with remarkable changes in their frequencies and/
with increasing temperatures. Initially, NPF-1 losses the physisorbed
◦
water molecules in the temperature range 47ꢀ 159 C. Thereafter, it is
chemically decomposed liberating the encapsulated oil components
followed by pyrolysis of organic residue at higher temperatures, be-
ꢀ 1
◦
◦
or strength. To name a few, the vibrational bands at 1647 cm (Δ
ν
=
tween 160 C and 489 C, as revealed from a significant weight loss.
These findings agree with the results of the previous work reported by
Hosseini, Zandi, Rezaei, and Farahmandghavi (2013) who reported that
the thermogram of the oregano essential oil (OEO)-loaded CSNPs dis-
played three-stages of weight loss. The 1st stage was observed in the
– –1
1
+
25), 859 & 754 cm , and 441 cm are attributed to the vibrations of
azomethine, imidazolium ring, and Zn–N groups of Zn(II)salen. On the
other hand, three main peaks observed at 2918, 2846, and 1621 are
assignable for the active components of the OSEO. The characteristic
peaks of amide II and β-glycoside groups of CSNPs for CSNPs can be seen
at 1549, 1243, and 1086, respectively. In addition, a strong stretch at
◦
temperature range 50 ꢀ 110 C and related only to the water evaporation
process. The 2nd and 3rd at higher temperature ranges were attributed
to dehydration of the saccharide moieties, degradation of chitosan
chain, decapsulation of OEO, and deacetylation of the biopolymer. On
the other hand, the Zn(II)Salen-encapsulated NPFs (NPF-2 and NPF-3)
showed almost similar TGA thermograms which are slightly different
from those of CSNPs and NPF-1 in that they are also thermally stable up
ꢀ
1
1
742 cm
(
νC=O) of Tween 20 and active components of the OSEO
confirms the sharing of Tween 20 in the nanoencapsulation of OSEO and
Zn(II)salen by CSNPs (Halevas et al., 2017).
3
.3.3. Thermal stability
◦
TGA offers a useful tool to investigate the thermal stability of any
to 50 C, however, they have exhibited three consecutive decomposition
new material, through studying the weight loss of a material as a
function of temperature. TGA thermograms of the as-prepared CSNPs
and its nanoplatforms (Fig. S4, ESIy) evident that CSNPs are thermally
stages at higher temperatures. These decomposition stages involve the
◦
liberation of physisorbed water (47ꢀ 160 C), de-capsulation of active
◦
constituents (161ꢀ 378 C), and pyrolysis of the residual organic maters
◦
◦
stable up to 50 C and then underwent two consecutive decomposition
(379ꢀ 493 C), respectively. The described tentative assignments are in
◦
steps under further heating. The first degradation step (50–163 C) in-
good agreement with the earlier reported thermogravimetric analysis
results (Duval, 1963; Halevas et al., 2017).
volves the departure of most physisorbed water molecules. In the second
Fig. 2. Zeta potential diagrams of (A) CSNPs, (B) OSEO@CSNPs (NPF-1), (C) Zn(II)Salen@CSNPs (NPF-2), and (B) Zn(II)Salen@OSEO@CSNPs (NPF-3).
7

Y.A. Hassan et al.
Carbohydrate Polymers 260 (2021) 117834
3
.3.4. Zeta potential
reported work by Yilmaz et al. (Yilmaz et al., 2019).
The zeta potential (ZP) of the bioactive material is a crucial param-
eter in the prediction of its surface charge that affects its physical sta-
bility in suspension and plays a significant role in its interaction with the
target site and consequently the material’s bioavailability. In this
context, the ZP of CSNPs and its different nanoplatforms were measured
and have been shown in Fig. 2. The values of ZP for all studied bioactive
nanoformulations ranged from +31.21 to +57.88 mV which are greater
than the minimum zeta potential (±30 mV) to obtain a physically stable
nanoparticles suspension (Glerieide et al., 2021), indicating that these
nanoplatforms are physically stable, by electrostatic repulsion, and have
no tendency to agglomerate. Interestingly, the Zn(II)Salen-encapsulated
NPFs have significantly higher ZP (P < 0.05) (+ 53.11 mV for NPF-2 and
3.3.5. Analysis of particle sizes
The particle size distributions of the bare CSNPs and its nanoplat-
forms (NBF-1, NBF-2, and NBF-2) were also investigated using the dy-
namic light scattering (DLS) technique (Fig. 3). As shown in Fig. 3A, the
diameters of CSNPs were in the range of 54.51–112.25 nm with poly-
dispersity indices (PDI) in the range of 0.29–0.35. On the other hand, the
particle size distribution constructed by the CSNPs after encapsulation of
OSEO was remarkably increased to be in the range of 69.42 nm (PDI
0.26) – 224.24 nm (PDI 0.35) (Fig. 3B). This may be attributed to the
hydrophobic interactions of the loaded EOs components which increase
the agglomeration of CSNPs (Yonar et al., 2021). Interestingly, the mean
diameter (MD) of CSNPs was significantly decreased after incorporation
of Zn(II)salen into its network (MD = 59.63 nm, PDI = 0.21) (Fig. 3C), in
comparison to bare CSNPs (MD = 95.25 nm) and NPF-1 (MD = 109.91
nm). This also could be ascribed to the electrostatic repulsion between
the cationic imidazolium terminals of salen complex which stabilizes the
Zn(II)Salen-loaded CSNPs (NPF-2). The average particle size of CSNPs
after encapsulation of both OSEO and Zn(II) complex was found to be
+
58.39 mV for NPF-3) than CSNPs (+32.58 mV). This increase may be
related to the spreading of cationic imidazolium groups into their
network (Elbehairi et al., 2020). The results also showed that the ZP
values of the OSEO-based NPFs, as well as their stability, have signifi-
cantly increased (P < 0.05) as compared to the native CSNPs. Conse-
quently, the solubility and dispersibility of OSEO in aqueous media have
also increased. Also, these results are consistent with the previously
Fig. 3. DSL of (A) CSNPs, (B) OSEO@CSNPs (NPF-1), (C) Zn(II)Salen@CSNPs (NPF-2), and (B) Zn(II)Salen@OSEO@CSNPs (NPF-3).
8

Y.A. Hassan et al.
Carbohydrate Polymers 260 (2021) 117834
1
20.15 ± 62.65 nm, whereas the PDI was in the range from 0.31 to 0.39.
OSEO are the hydrophobic interactions of the loaded EOs components
and swelling of the CSNPs owing to the layering of OSEO components on
their surfaces (Hosseini et al., 2013). On the other hand, the tendency of
CSNPs for agglomeration was decreased after encapsulation of Zn(II)
salen as evidenced in Fig. 4C. This may be ascribed to the formation of
an electrostatic repulsion layer by the cationic imidazolium compart-
ments on the surface of CSNPs. This nanoformulation (NPF-2) is
composed of spherical NPs of average size 49.56–157.12 nm. Contrary,
the average size of CSNPs was increased to reach 61.34–215.28 nm after
loading of both OSEO and Zn(II)salen. This may be attributed to the
occupation of the hydrophobic interior of CSNPs with OSEO particu-
lates, pushing the hydrophilic Zn(II)salen molecules to the hydrophilic
sites of CNPs, thereby resulting in an increase of its average diameter
(Halevas et al., 2017).
Noteworthy, low values of PDI for CSNPs and its NPFs are indicative of a
narrow-size distribution of these nanoformulations in their respective
suspension, and consequently, a homogeneous dispersion is obtained,
which is useful for pharmacological applications.
3
.3.6. Scanning electron microscope (SEM)
The morphological features of the surfaces of CSNPs and its nano-
platforms were inspected using the SEM technique. The SEM micro-
graphs of the inspected samples were shown in Fig. 4. The surface of
CSNPs sample displayed semi-spherical NPs, predominantly agglom-
erate in assemblies of a spongy shape and high density (Fig. 4A). This
spongy nature for CSNPs provides them active accessible cavities for
effective encapsulation of OSEO and Zn(II)salen. The SEM images
revealed that the average size of CSNPs is about 67.21–129.17 nm, with
a narrow size distribution. As shown in Fig. 4A, the texture of OSEO-
loaded CSNPs (NBF-1) exhibits spherical NPs assembled in bulky ag-
gregates with an average diameter of 75.42–233.24 nm. Again, the
possible reasons for increasing the average size of CSNPs after loading of
3
.4. Loading capacity and encapsulation efficiency
The in situ loading capacity (LC) and encapsulation efficiency (EE) of
the CSNPs-based nanoplatforms samples were estimated using atomic
Fig. 4. SEM micrographs of (A) CSNPs, (B) OSEO@CSNPs (NPF-1), (C) Zn(II)Salen@CSNPs (NPF-2), and (D) Zn(II)Salen@OSEO@CSNPs (NPF-3).
9

Y.A. Hassan et al.
Carbohydrate Polymers 260 (2021) 117834
absorption and UV–vis for Zn(II)Salen and OSEO nanoformulations,
3.5.1. In vitro OSEO release study
respectively. The obtained results are as follow;
Generally, the release of active components from nanocapsules or
microcapsules can be induced through multiple mechanisms including;
surface erosion, biodegradation, disintegration, desorption, and diffu-
sion (Halevas et al., 2017). The in vitro release profiles of OSEO from
OSEO-loaded CSNPs–Tween 20 nanoplatforms (NPF-1, NPF-3) were
studied at phosphate buffer saline (pH 7.4) for 48 h and the percentages
of OSEO released from the total encapsulated OSEO content with time
are represented in Fig. 5A. The OSEO release pattern displayed a
biphasic process including an initial burst phase followed by a very slow
OSEO release rates phase and finally reaching a plateau. During the first
6 h, the initial burst release of OSEO was significantly increased (P <
0.05) to reach the release percent of 67.8 % and 77.1 % for NPF-1 and
NPF-2, respectively (Fig. 5A). The burst release of OSEO at this stage was
attributed to the OSEO molecules adsorbed on and encapsulated near
the surface of the nanoparticles, as the leaching rate of the chitosan near
the surface is high and thus OSEO released would also be high (Hosseini
et al., 2013). In the second stage, the release rate was significantly
decreased (P < 0.05) and the percent of cumulative OSEO release
reached 72.9 and 83.2 % for NPF-1 and NPF-3, respectively, after 24 h.
Thereafter, almost steady release profiles have been attained from 24 h
to 48 h (Fig. 5A). This decrease can be ascribed to the diffusion of
dispersed OSEO into the matrix of CSNPs which leads to slow down the
release rate of OSEO from its encapsulated forms (Esmaeili & Asgari,
2015). In addition, the co-encapsulation of OSEO and Zn(II)Salen with
CSNPs in the interior of Zn(II)Salen@CMEO-loaded CSNPs leads to a
relatively sharp rise of the OSEO release rate compared to the
OSEO-loaded sample as revealed from the elevation of the OSEO release
percentage from Zn(II)Salen@OSEO-loaded CSNPs in comparison to
OSEO-loaded CSNPs. This behavior can be attributed to the hydrophilic
nature of the inner network of CSNPs that promotes the adsorption of Zn
3
.4.1. OSEO loading capacity (LC) and encapsulation efficiency (EE)
The content of OSEO (loading capacity) in the OSEO-loaded and Zn
(
II)Salen@OSEO-loaded CSNPs (NBF-1 and NBF-3) were quantified to
be 20.98 % and 13.92 %, respectively, whereas the OSEO encapsulation
efficiency by CSNPs for the same nanoformulations were found to be
2
8.28 % and 19.72 %, respectively. The elevated LC values can be
attributed to the incorporation of a high initial concentration of OSEO
during the synthesis of CSNPs, whereas, the relatively low EE may be
ascribed to the saturation of the CSNPs network with the loading OSEO
particles, and the excess OSEO was adsorbed only on the surface of NPs.
This finding was in agreement with previous reports. These findings are
consistent with previous reports (Ajun, Yan, Li, & Huili, 2009; Hosseini
et al., 2013; Kavaz, Idris, & Onyebuchi, 2019; Su et al., 2020; Yoksan,
Jirawutthiwongchai, & Arpo, 2010).
3
.4.2. Zn(II)Salen loading capacity and encapsulation efficiency
The LC of Zn(II)Salen in the NBF-2 and NBF-3 were estimated to be
3.34 % and 8.49 %, respectively, whereas the EE of Zn(II)Salen in the
1
same samples were 29.78 % and 15.45 %, respectively. The relatively
satisfying LC and EE percentages for nanoformulations indicate the
adequacy of the CSNPs as nanocapsules for a cocktail of metal-complex
and EO. However, the simultaneous encapsulation of both complex and
EO into the matrix of CSNPs reduces the loading capacity and encap-
sulation efficiency for each component, as compared to the encapsula-
tion of each component separately. The difference in the results of EE for
OSEO and Zn(II)Salen can be related to the variation of the chemical
features for the active compound to be encapsulated (da Rosaa et al.,
2
020).
(
II)Salen into the core of CSNPs, whereas, the hydrophobic nature of
CSNPs surface, owing to amphiphilic Tween 20, makes more OSEO
molecules are adsorbed on or entrapped near the surface of the CSNPs
compared to OSEO-loaded CSNPs. These results are indicative of the
suitability of CSNPs-Tween 20 nanosystems as Nano-vehicles to control
delivering of a cocktail from EO with the metal complex.
3
.5. In vitro release studies
The release profile of the bioactive ingredients OSEO and Zn(II)Salen
from chitosan nanocapsules is a crucial pharmacokinetic parameter in
the accreditation of the suitability of using CSNPs as nanocapsules for
these ingredients. The pharmacokinetic behavior and the release profile
of OSEO-loaded and Zn(II)Salen-loaded CSNPs is strongly-correlated to
the total amount of OSEO-loaded or Zn(II)Salen released and the rate of
its releasing with time. The obtained results clearly demonstrate that the
current approach offers a facile method for simultaneous encapsulation
of Zn(II) complexes and EOs in the biocompatible CSNPs.
3
.5.2. In vitro Zn(II)Salen release study
As shown in Fig. 5B, the in vitro release of Zn(II)salen from Zn(II)
Salen-loaded (NPF-2) and Zn(II)Salen@ OSEO-loaded CSNPs (NPF-3)
was also proceed via a two-step biphasic process. The initial burst release
stage was continued for the first 6 h to record the cumulative Zn(II)
Fig. 5. (A) Cumulative released OSEO percentage with regard to the total entrapped OSEO vs. time, for CMEO-loaded CSNPs (NPF-1) and Zn(II)Salen@CMEO-loaded
CSNPs (NPF-3). (B) Cumulative released Zn(II)salen percentage with regard to the total entrapped Zn(II)salen vs. time, for Zn(II)Salen-loaded CSNPs (NPF-2) and Zn
(
II)Salen@CMEO-loaded CSNPs (NPF-3).
1
0

Y.A. Hassan et al.
Carbohydrate Polymers 260 (2021) 117834
release of percentages 81.7 and 92.3 % for NPF-2 and NPF-3, respec-
tively. This burst release of Zn(II)salen from NPF-3 may be ascribed to;
be attacked by the bioactive components of the EO (Burt, 2004). To
name a few, EO components can damage the structure of the bacterial
cell wall and its cytoplasm membrane by interaction with their active
constituents (polysaccharides, fatty acids, and phospholipids layers)
(Burt, 2004). In addition, the bioactive ingredients of EO can induce
enzyme inhibition and interfere with the energy generation system
(ATPase), preventing the bacterial cell to produce the energy essential
for its survival (Speranza & Corbo, 2010).
(
i) the higher content of Zn(II)salen molecules adsorbed on and
entrapped near the surface of CSNPs, due to OSEO inclusion competi-
tive, enabling them to directly contact the releasing medium and
consequently more facile to release, (ii) higher surface erosion rate of
the CSNPs owing to the presence of hydrophilic Tween 20 moieties, and
(
iii) the hydrophilic nature of the Zn(II)salen molecules due to imida-
zolium terminals, resulting in an increased dissolution rate in the
ambient release medium. In the second stage, the release rate was
significantly decreased (P < 0.05) and the percent of cumulative Zn(II)
salen release reached 90.7 and 98.2 % for NPF-2 and NPF-3, respec-
tively, after 24 h. Thereafter, almost steady release profiles have been
attained from 24 h to 48 h (Fig. 5B). Again, the obtained results clearly
refer to the suitability of utilizing the biocompatible CSNPs as facile and
effective tools of simultaneous nanoencapsulation of EO with a metal
complex.
The results of antifungal activity of OSEO (Fig. S5, ESI†) demon-
strated that the OSEO ingredients are more active as bactericidal than as
fungicidal. In addition, the inhibition of fungal growth occurs with a
species-dependent performance. The PI fungus is the most sensitive to-
ward the oil components (ZOI 23.9 ± 1.4 mm), whereas, CA is the most
resistant (ZOI 16.1 ± 1.1 mm). The MIC values of OSEO against exam-
ined bacterial fungal species are in the range of 2.15–6.45
μL/mL. The
antifungal activities seem to be in accordance with previous reports
indicating that the essential oils, rich in phenolic compounds, possess
high levels of bioactivity (Bouchra, Achouri, Hassani, & Hmamouchi,
2003; El Gendy et al., 2015; Lambert, Skandamis, Coote, & Nychas,
3
.6. In vitro antimicrobial studies
2
001). Generally, the antifungal activity of oregano EOs is mostly due to
the presence of phenolic compounds (thymol and carvacrol) among its
constituents (Portillo-Ruiz, Ramos,
S ´a nchez, Mu n˜ oz,
The antimicrobial potencies of the OSEO, Zn(II)Salen, CSNPs, and
nanoplatforms has in vitro assessed in comparison to standard antimi-
crobial drugs (Penicillin G (Pen G) (C16 S, 334.39 g/mol) for
Gram-positive bacteria, Streptomycin (ST) (C21 12, 581.57 g/
mol) for Gram-negative bacteria, and Clotrimazole (CLOT) (C22 17ClN
44.84 g/mol) as an antifungal one). The recorded values of the diam-
eter of zone of inhibition (ZOI) and minimum inhibitory concentration
MIC) against tested microorganisms have been used as antimicrobial
indices.
&
H
18
N
2
O
4
Nev a´ rez-Moorill o´ n, 2012). These active ingredients have the capacity to
induce several mechanisms in the fungal cell such as; (i) interference
with the activity of cell wall enzymes like chitin synthase/chitinase as
39 7
H N O
H
2
,
3
well as
α- and β-glucanases of fungal celli, altering its permeability and
permitting the loss of active constituents of the cell (Adams, Kunz, &
Weidenb o¨ rner, 1996), (ii) interaction with the membrane proteins,
inducing malfunction of their structure and functionality (Fung, Taylor,
(
&
Kahan, 1977).
3
.6.1. Antimicrobial performance of OSEO
The diameters of ZOI induced by the free OSEO into different culti-
3.6.2. Antimicrobial performance of Zn(II)Salen
vated tested microorganisms were measured using the agar well diffu-
sion method and the results have presented in Fig. S5 (ESIy). The
obtained results revealed the native OSEO exhibited significant and
broad-spectrum antimicrobial potencies against all tested microorgan-
isms, with performance depending on the microbial species and oil
concentration. In general, OSEO inhibited the growth of Gram-positive
strains slightly more effectively than Gram-negative strains. Gram-
positive bacteria are more sensitive toward OSEO in comparison to
Gram-negative ones. For instance, the diameters of ZOI of the OSEO
against SA and BS were found to be 23.22 ± 1.68 and 22.21 ± 1.45 mm,
respectively; whereas, the inhibition zones obtained against EA and PA
were 21.42 ± 1.59 and 18.64 ± 1.21 mm, respectively. These findings
are consistent with the previous study performed by Gendy et al. (El
Gendy et al., 2015). The difference in sensitivity of the bacterial strains
toward OSEO is thought to arise owing to the differences in their
respective cell wall structure. Gram-negative bacteria have more com-
plex cell envelopes than Gram-positive species. The cell wall of the
Gram-negative bacteria is composed of two protective layers that pro-
vide it rigidity: (i) A thin peptidoglycan layer and (ii) An outer mem-
brane comprise an exterior lipopolysaccharide (LPS) layer and a
phospholipids layer in the interior. On the other hand, the Gram-positive
bacteria have only one protective layer (a thick peptidoglycan layer) but
lack the outer membrane. Therefore, the oil active components can
diffuse easily through the loose outer wall of Gram-positive bacteria,
making them more susceptible to the action of bioactive components of
EO (Angienda, Onyango, & Hill, 2010). Noteworthy, the antibacterial
effect has significantly enhanced with an increase in the concentration
of the OSEO. For example, the largest ZOI was obtained against S. aureus
The ZOI values of Zn(II)Salen complex (Fig. S6, ESIy) revealed its
higher antimicrobial potencies, compared to OSEO and Pen G, against
the Gram-negative bacteria (EC, PA), whereas, it was demonstrated to be
less active against the Gram-positive bacteria (SA, BS), in comparison to
OSEO. Specifically, Zn(II)Salen had ZOIs values in the range of
(19.35–26.12 mm) which is higher than that of OSEO (ZOI 18.64–23.22
mm), and Pen G (16.71–20.98 mm). The MIC values of Zn(II)Salen were:
16.44, 29.48, 4.03, and 53.65 g/mL against SA, BS, EC, and PA mi-
μ
croorganisms, respectively. Consequently, co-encapsulation of Zn(II)
salen with OSEO could offer a promising antimicrobial cocktail to
effectively and synergistically treat both types of bacterial infections.
The in vitro antifungal study of Zn(II)Salen demonstrated that the Zn
(II) complex has satisfiable antifungal activity as revealed from the ZOI
values (AF, 22.63 ± 0.89; CA, 21.93 ± 0.87 mm) which are comparable
to or exceed that of the standard antifungal drug (CLOT) (AF, 22.49 ±
0.79; CA, 18.09 ± 0.91 mm). The MIC values of Zn(II)Salen were: 52.58
and 55.67 g/mL against AF and CA microorganisms, respectively.
μ
3.6.3. Antimicrobial performance of CSNPs and its nanoplatforms
The synergistic antibacterial potencies of Zn(II)Salen, OSEO, and Zn
(II)Salen@OSEO cocktail were further investigated after encapsulation
with CSNPs. The antibacterial activity of each nano- formulation was
studied with three serial concentrations (25, 50, and 100 g/mL), the
μ
findings (ZOI values) were collected in Fig. 6. In general, the antimi-
crobial activity of the OSEO has remarkably enhanced after encapsula-
tion with CSNPs, as compared to the native OSEO and CSNPs
components. Where the ZOI values of OSEO-encapsulated CSNPs (NPF-
1) were; 29.48 ± 1.26, 27.42 ± 0.99, 24.31 ± 1.05, and 18.89 ± 0.98
against SA, BS, EC, and PA microorganisms, respectively, which are
higher than that recorded for the native OSEO (ZOI 18.64–23.22 mm)
and CSNPs (ZOI 10.31–13.12 mm). Meanwhile, the MIC values of NPF-1
were decreased in comparison to bare OSEO (MICNPF-1/OSEO values;
0.75/1.25, 1.35/2.15, 2.15/2.75, and 3.5/3.98 toward SA, BS, EC, and
PA, respectively). Thus, OSEO was found to have a synergistic
(
23.22 ± 1.68 mm) with 4
ZOI has diminished to (16.4 ± 1.32 mm) at 1
MIC values of OSEO against tested bacterial strains are in the range of
L/mL. Due to the variety of chemical compounds present in
μ
L/well concentration, whereas, the value of
μL/well concentration. The
1
.25–3.98 μ
the EO, their antibacterial potencies could not be related to one specific
mechanism, but there are several targets of the bacterial cell that might
1
1

Y.A. Hassan et al.
Carbohydrate Polymers 260 (2021) 117834
Fig. 6. Graph for ZOI values for CSNPs and its new nanoplatforms as function in sample concentration and bacterial species (A) S. aureus (SA, RCMB 000106), (B)
B. subtilis (BS, RCMB 000101), (C) E. coli (EC, RCMB 000103), and (D) P. aeruginosa (PA, RCMB 000102).
antibacterial action with CSNPs. In a similar fashion, Zn(II)Salen-loaded
CSNPs (NPF-2) displayed greater antimicrobial efficacy than the nascent
Zn(II)Salen complex. The ZOIs values of NPF-2 against bacterial species
have elevated to be in the range of 26.75–29.95 mm that higher than the
range recorded for nascent Zn(II)salen (19.35–26.12 mm). Furthermore,
fungal species.
In summary, the new antimicrobial agents (OSEO and Zn(II)Salen),
as well as the encapsulating agent (CSNPs), contributed to different
degrees in the synergistic antimicrobial actions for the novel nano-
formulations. Furthermore, the involvement of both OSEO and Zn(II)
Salen in a single nanoplatform (NPF-3) is synergistically enhanced the
antimicrobial effect as indicated from, despite, the lowering content of
OSEO and Zn(II)Salen from 20.93 % and 29.78 in NFP-1 and NPF-2,
respectively, into 13.92 % and 15.45 % in NPF-3, however, the MIC
values have significantly decreased. For instance, the MIC values of NPF-
NPF-2 (MIC; 3.25–45.25
μ
g/mL) exhibited lower MIC values than that
assigned for free Zn(II)Salen (MIC; 4.03–53.65
μ
g/mL). Therefore, also,
a combination of Zn(II)salen and CSNPs displayed a synergistic anti-
bacterial effect.
Accordingly, when investigating the antimicrobial properties of Zn
(
II)salen@OSEO-loaded CSNPs (NPF-3), it would be expected it
exhibit greater antimicrobial potencies in comparison to the nascent Zn
II)salen, OSEO, and CSNPs components. In that respect, the ZOI and
1, NPF-2, and NPF-3 toward EC were found 2.15, 3.25, and 0.25 g/mL,
μ
respectively.
(
The novelty of the present study can be highlighted in the following
points; (i) fabrication, for the first time, a new generation of nano-
pharmaceuticals incorporating several pharmacophore cocktails (EO,
Metallo-Schiff base, imidazolium ionic liquid, and CSNPs). (ii) the
elevated positive ZP values for our new nanoplatforms (48.17–58.39
mV), and thus great stability, as compared to previously reported similar
nanoformulation (tea polyphenol (TP)-Zn complex loaded CSNPs
nanoformulation (39.17–45.62 mV)) (Zhang & Zhao, 2015). (iii) The
smaller particle size (57.5–182.77 nm) and PDI values (0.31–0.39) in
comparison to those in the literature (Lin et al., 2015; Rodriguez et al.,
2019; Zhang & Zhao, 2015) are very important indices for promoting
their antibacterial activity to great extent.
MIC values of NPF-3 were significantly enhanced against different tested
microbes. The ZOI values of NPF-3 were in the range of 32.55–35.75
mm, whereas, its MIC values were ranged from 0.25 to 2.50 g/mL. The
μ
amazing antimicrobial effects of Zn(II)salen@OSEO-loaded CSNPs
NPF-3) is attributed to the synergistic antibacterial actions of its
(
bioactive ingredients (Zn(II)salen, OSEO, and CSNPs).
Furthermore, in general, the antifungal activity for all types of
CSNPs-based nanoplatforms has remarkably enhanced as compared to
the individual components of each nanoplatform. In that respect, the
ZOI values of examined nanoplatforms (NPF-1, NPF-2, and NPF-3) have
decreased to be in the range of 21.25–27.50 mm, whereas, the MIC
values were ranged from 12.25
μ
g/mL to 50.25 g/mL, against all tested
μ
1
2

Y.A. Hassan et al.
Carbohydrate Polymers 260 (2021) 117834
4
. Conclusions
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The facile protocols have been designed to prepare new antimicro-
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(
(
OSEO), from Origanum Syriacum L. plant, shrimp chitosan nanoparticles
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used as synergistic encapsulating and antimicrobial agents for sepa-
rately or co-encapsulation of OSEO and Zn(II)Salen; targeting fabrica-
tion of extremely potent antimicrobial nanoplatforms "cocktails" (OSEO-
loaded CSNPs (NPF-1), Zn(II)Salen-loaded CSNPs (NPF-2), and Zn(II)
Salen@OSEO-loaded CSNPs (NPF-3)). All fabricated materials were
structurally and morphologically characterized. The acceptable loading
capacity (LC), encapsulation efficiency (EE), and in vitro release profiles
of OSEO and Zn(II)Salen in their respective nanoplatforms are indicative
of the suitability of using CSNPs as nanocapsules for a cocktail of Zn(II)
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The extensive step-by-step antimicrobial studies have been carried
out against a common panel of pathogenic microbes (bacteria and
fungi). The findings of this study could be drawn as follow; (i) OSEO and
Zn(II)Salen have remarkable capacities to curb the growth of all tested
microbial strains. (ii) The antimicrobial activities of the OSEO and Zn(II)
Salen have significantly enhanced after encapsulation with CSNPs, as
revealed from the ZOI and MIC values. (iii) All the tested materials are
more active as bactericidal than as fungicidal. (iv) The Gram-positive
bacteria (SA and BS) are more sensitive than Gram-negative ones (EC
and PA) toward OSEO and its nanoplatforms (NPF-1 and NPF-3),
whereas, the vice versa effects have been observed for Zn(II)Salen com-
plex and its nanoplatforms (NPF-1 and NPF-3).
In general, the amazing physicochemical features of new nanoplat-
forms and their bioactive ingredients (Zn(II)Salen, OSEO, and CSNPs)
signify the importance of our designs for developing a new generation of
nanopharmaceuticals supported both natural products and biogenic
ionic metal cofactors, targeting the multidrug resistant (MDR)
pathogens.
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CRediT authorship contribution statement
biopolymer as a matrix. Pharmaceutics, 12, 669.
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Environmental Chemistry Letters, 16, 101–112.
Yasser A. Hassan: Methodology, Data curation, Software, Valida-
tion, Visualization, Writing - original draft. Amgad I.M. Khedr: Meth-
odology, Software, Data curation, Validation, Visualization, Writing -
original draft, Writing - review & editing. J. Alkabli: Methodology,
Conceptualization, Data curation, Writing - original draft. Reda F.M.
Elshaarawy: Conceptualization, Methodology, Data curation, Valida-
tion, Visualization, Writing - original draft, Writing - review & editing.
Ali M. Nasr: Methodology, Data curation, Software, Validation, Visu-
alization, Writing - original draft.
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Amsterdam: Elsevier Publishing Co.
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cultivated Origanum.
Elbehairi, S. E. I., Ismail, L. A., Alfaifi, M. Y., Elshaarawy, R. F. M., & Hafez, H. S. (2020).
Chitosan nano-vehicles as biocompatible delivering tools for a new Ag(I)
curcuminoid-Gboxin analog complex in cancer and inflammation therapy.
International Journal of Biological Macromolecules, 165(B), 2750–2764.
Elshaarawy, R. F. M., & Janiak, C. (2016). Antibacterial susceptibility of new copper (II)
N-pyruvoyl anthranilate complexes against marine bacterial strains–In search of new
antibiofouling candidate. Arabian Journal of Chemistry, 9(6), 825–834.
Elshaarawy, R. F. M., Mustafa, F. H. A., van Geelen, L., Abou-Talebe, A. E. A.,
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polyelectrolyte chitosan anti-biofoulants: Fabrication of high-performance economic
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.carbpol.2021.117834.
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