Polyphosphonium-oligochitosans decorated with nanosilver as new prospective inhibitors for common human enteric viruses

Article abstract of DOI:10.1016/j.carbpol.2019.115261

The main objective of this work to explore new safe antiviral agents against hepatitis A virus (HAV), norovirus (NoV) and Coxsackievirus B4 (CoxB4) infections. In this context, we have successfully prepared new polyquaternary phosphonium oligochitosans (P

Full text of DOI:10.1016/j.carbpol.2019.115261

Carbohydrate Polymers 226 (2019) 115261
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Polyphosphonium-oligochitosans decorated with nanosilver as new
prospective inhibitors for common human enteric viruses
a,⁎
a
b
c
Ahmed R. Sofy , Ahmed A. Hmed , Naglaa F. Abd El Haliem , Mohamed A.-E. Zein ,
d,e,⁎⁎
Reda F.M. Elshaarawy
Botany and Microbiology Department, Faculty of Science, Al-Azhar University, 11884 Nasr City, Cairo, Egypt
Microbiology and Immunology Department, Faculty of Medicine (Girls), Al-Azhar University, Nasr City, Cairo, Egypt
Faculty of Science, Chemistry Department, Damanhour University, Egypt
Institut für Anorganische Chemie und Strukturchemie, Heinrich-Heine Universität Düsseldorf, 40204 Düsseldorf, Germany
Faculty of Science, Suez University, Suez, Egypt
a
b
c
d
e
A R T I C L E I N F O
A B S T R A C T
Keywords:
The main objective of this work to explore new safe antiviral agents against hepatitis A virus (HAV), norovirus
(NoV) and Coxsackievirus B4 (CoxB4) infections. In this context, we have successfully prepared new poly-
quaternary phosphonium oligochitosans (PQPOC1,2) to use them as natural synergistic in-situ bioreductants of
silver ions into nanosilver and stabilizing agent for these nanosilver to fabricate PQPOCs-AgNPs nano-bio-
composites (NBC1,2). The antiviral performance of the PQPOCs and NBCs against FCV, HAV, and CoxB4 reflects
great virucidal activities for NBCs as compared with PQPOCs with maximum viral reduction% (41.42, 80.62, and
Polyphosphonium oligochitosans
Nanosilver-based biocomposites
Antiviral
Enterovirus replication inhibitors
84.04%) for NBC1 against FCV, HAV, and CoxB4, respectively. Furthermore, the antiviral activity of NBC1 is
concentration- / pH-dependent where NBC1 acquired its maximum antiviral at [NBC1] = 200 μL/mL and pH 4.
Based upon these facts, we could attribute the enhanced virucidal efficacy of NBC1: (i) binding of AgNPs to the
virions active sites. (ii) Electrostatic interaction between the positive brushes of PQPOC and negative targets of
viruses. (iii) Inducing ribonuclease catalyzed by CS to degrade the viral RNA and consequently prevents its
transcription and translation.
1. Introduction
enhance the quality and safety of food products but also to serve as
natural antivirals (Randazzo, Fabra, Falcó, López‐Rubio, & Sánchez,
Enteric viruses play a pivotal role in food-/water-borne diseases
FBDs/ WBDs) outbreaks (Lodder, van den Berg, Rutjes, & de Roda
2018).
Amongst natural biopolymers, the inherent biodegradability, bio-
(
Husman, 2010; Prevost et al., 2015). Among these FB/WB viruses,
hepatitis A virus (HAV) and enterovirus (EV) such as human norovirus
compatibility, antimicrobial, antiviral and antibiofilm efficacies, non-
toxicity and eco-friendship of chitosan (CS) biopolymer (Randazzo
et al., 2018; Elshaarawy, Refaee, & El-Sawi, 2016; Guan et al., 2019)
make it a promising scaffold for constructing of new smart pharmaco-
logical materials. Nevertheless, the widespread application of native CS
as a pharmacological agent is restricted due to its poor aqueous solu-
bility and thus limited bioavailability, low thermal and mechanical
stability. Surface-functionalization of CS acts as the magic solution to
tackle these challenges (Elshaarawy, Mustafa, Herbst, Farag, & Janiak,
2016, 2018). Notably, our earlier outputs (Elshaarawy, Mustafa et al.,
2016; Elshaarawy & Janiak, 2014; Elshaarawy et al., 2017) revealed
that decoration of the pharmacological molecules with ionic liquid (IL)
(
NoV) and Coxsackievirus B4 (CoxB4) have attracted the interest of
numerous researchers due to their serious impacts on public health and
environment (Sofy et al., 2018a, 2018b; Haas, Rose, Gerba, & Regli,
1993; EFSA, 2016). Globally, it was found that 120 million FBDs were
caused by viral contamination of food, annually (WHO, 2015). Most of
these viruses have no licensed antiviral drug. Thus, there is an im-
perative need for exploring new effective and safe therapeutics for these
FB viruses, particularly for NoV. In this context, a great interest was
directed toward natural compounds as antiviral candidates. Natural
biopolymers potentially have synergistic multifunctions, not only to
⁎
Corresponding author.
⁎
⁎
Corresponding author at: ARS, Botany and Microbiology Department, Faculty of Science, Al-Azhar University, 11884 Napa City, Cairo, Egypt; RFME, Institut für
Anorganische Chemie und Strukturchemie, Heinrich-Heine Universität Düsseldorf, 40204 Düsseldorf, Germany.
E-mail addresses: ahmed_sofy@azhar.edu.eg (A.R. Sofy), reel-001@hhu.de, reda.elshaarawy@suezuniv.edu.eg (R.F.M. Elshaarawy).
https://doi.org/10.1016/j.carbpol.2019.115261
Received 10 June 2019; Received in revised form 20 July 2019; Accepted 27 August 2019
Available online 28 August 2019
0144-8617/ © 2019 Elsevier Ltd. All rights reserved.

A.R. Sofy, et al.
Carbohydrate Polymers 226 (2019) 115261
motifs resulting in synergetic effects of amelioration the aqueous so-
lubility and significant enhancement of the antimicrobial performance.
Through the last few decades, silver nanoparticles (AgNPs) have
become in the forefront of metal NPs-based biomedical materials be-
cause of their unmatched properties including ease of the fabrication
with low cost, great surface area and remarkable broad-spectrum an-
timicrobial activities (Durán, Nakazato, & Seabra, 2016). Thus, AgNPs-
based materials always offered interesting, challenging, and promising
characteristics suitable for diverse applications. Notablly, AgNPs-based
antiviral agents are incredibly useful in combating many viruses such as
Adenovirus (Chen, Zheng, Yin, Li, & Zheng, 2013), Bovine herpesvirus-
2.1.1. Preparation of PQPOCs
Generally, a 25 ml ethanolic solution of TPP salts (2a,b) (equivalent
to molar NH -content in OC which can be calculated from N% in OC)
2
was added dropwise to a 75 mL of a 50% (v/v) mixed-solvent (2%
AcOHaq/ EtOH) solution containing 1 g of OC under vigorous stirring at
70 °C. Then the solution was stirred for more 24 h at the same condi-
tions. Thereafter, the reaction mixture was concentrated under reduced
pressure to give a gel-like residue which diluted with an excessive
amount of ethyl acetate (AcOEt) and ultrasonically irradiated for 1 h to
remove the solvent and solidify these gel-like products. The isolated
solids were filtered and washed with EtOH:AcOEt mixtures (30:70,
20:80, and 0:100 V/V, sequentially). Finally, the desired products
PQPOC1,2 were dried at 35 °C under vacuum for 24 h and characterized
as follows;
1
(El-Mohamady, Ghattas, Zawrah, & Abd El-Hafeiz, 2018), Coxsack-
ievirus B3 (Shaheen, El-hadedy, & Ali, 2019), Chikungunya (Sharma
et al., 2019), Hepatitis B (Lu et al., 2008), Human parainfluenza type 3
(
&
Gaikwad et al., 2013), Human immunodeficiency virus type 1 (Trefry
Wooley, 2013), Influenza A (Park et al., 2018; Park et al., 2018), and
Poly-N-(((3-(3-hydroxy-4-(1-iminoethyl)phenoxy)propyl)triphe-
nylphosphonium chloride) oligochitosan (PQPOC
1
): Canary yellow
powder, Yield (1.72 g). ATR-FTIR (cm ): 3384 (m, br, ν(O-H + NH2)),
3229 (m, sh, ν N-H)), 3096 (m, br, ν(Ar-H)), 2956 (m, br, ν(C-H)), 1638 (vs,
sh, ν(C=O + C=N)), 1588 (vs, sh, ν(amide II)), 1537 (m, sh), 1454 (s, sh,
(Ar-P)), 1382 (s, sh, ν(amide III)), 1275 (m, sh, ν(Ar O)), 1112 (s, sh, ν(Ar-
P+Cl–)), 1082 (s, sh), 893 (s, sh ν(C-O-C), β-glycosidic linkage), 736 (s, sh).
−1
Monkeypox (Rogers, Parkinson, Choi, Speshock, & Hussain, 2008).
Also, AgNPs may block viral receptors and/or interfere with the viral
genome when a virus enters the host cells, by inactivating viral particles
outside the host cells (Khandelwal, Kaur, Kumar, & Tiwari, 2014).
Unfortunately, the widespread biomedical applications of AgNPs are
restricted by their hygienic and environmental problems (Leon-Silva,
Fernandez-Luqueno, & Lopez-Valdez, 2016). Whereas the leaching of
Ag(I) ions from the surface of AgNPs (Gaillet & Rouanet, 2015) and
their agglomeration into bulky particles or fibers, may fluctuate their
biological features and cause serious impacts upon the environment and
the human health (Ray, Yu, & Fu, 2009). Therefore, exploring novel,
innovative strategies for enveloping and stabilization of AgNPs is an
urgent need to overcome these obstacles.
(
ν
-
1
3 2
H NMR (600 MHz, 1% CD COOD/D O)60ºC δ (ppm): 10.27 (s, 2H, 2 x
O-H), 8.21 (s, 1H, N-H), 7.72 (d, J =7.0 Hz, 2H, 2 x Ar-H), 7.65 (d, J
=7.1 Hz, 12H, 12 x Ar-H), 7.58-7.39 (m, 12H, 12 x Ar-H), 7.35-7.18
(m, 12H, 12 x Ar-H), 6.87 (d, J =6.8 Hz, 2H, 2 x Ar-H), 6.58 (s, 2H, 2 x
Ar-H), 5.63 (s, br, 4H, chitosan-H), 4.26 (t, J =1.5 Hz, 4H, 2 x Ar-O-
CH ), 4.18-3.91 (m, 12H, chitosan-H), 3.83 (s, br, 4H, chitosan-H), 3.75
2
(t, J =7.0 Hz, 4H, chitosan-H), 3.66 (s, br, 4H, chitosan-H), 3.56-3.35
(m, 8H, chitosan-H), 3.05 (t, J =6.9 Hz, 4H, chitosan-H), 2.51 (t, J
+
Despite extensive work has been reported to prepare AgNPs using
CS as synergistic reducing, enveloping and stabilizing agents (Bui, Park,
&
=1.4 Hz, 4H,
CH CH CH
NHAc), 1.82 (s, 6H, 2 x CH
2
x
P-CH
2
CH
2
), 2.22-2.08 (m, 4H,
2
x
Ar-O-
), 1.84 (s, 3H,
), 1.32-1.18 (m, 4H, 2 x Ar-O-CH CH CH ).
COOD/D O)60ºC δ (ppm): 175.68 (C = O),
+
2
2
2 2 2 2
), 1.96-1.86 (m, 4H, 2 x P-CH CH CH
Lee, 2017; Kalaivani et al., 2018; Murugadoss & Chattopadhyay,
008), however, no attention has been paid toward the utilization of
3
2
2
2
13
2
C NMR (151 MHz, 1% CD
3
2
poly quaternary phosphonium (PQP)-based CS as a synergistic re-
ductant and capping agent. Noteworthy that the triphenylphosphonium
(
structural features which enable them to form delocalized lipophilic
cations (Wang, Shao, Zhang, & Cheng, 2014; Wang, Xu, Zhao, & Zhao,
2
hydrophobic environment and selectively accumulate in the mi-
tochondria of pathogenic cells because of their higher trans-membrane
potentials in comparison to the normal cells (Biswas, Dodwadkar,
164.79 (C = N), 163.81 (phenoxy C-O), 162.05 (phenolic C-OH),
136.43 (Ar-C), 132.13 (Ar-C), 131.09 (Ar-C), 129.96 (Ar-C), 118.77
(Ar-C), 117.32 (Ar-C), 112.10 (Ar-C), 109.71 (Ar-C), 109.65 (Ar-C),
108.41 (chitosan-C), 105.14 (chitosan-C), 87.78 (chitosan-C), 82.89
(chitosan-C), 82.78 (chitosan-C), 81.83 (chitosan-C), 81.71 (chitosan-
C), 80.23 (chitosan-C), 79.69 (chitosan-C), 78.93 (chitosan-C), 75.91
(chitosan-C), 75.08 (chitosan-C), 71.95 (chitosan-C), 71.45 (chitosan-
TPP)-based quaternary phosphonium salts (QPS) possess unique
014). Also, TPP can preferentially migrate from an aqueous to the
C), 68.93 (Ar-O-CH
(chitosan-C), 62.88 (chitosan-C), 61.82 (chitosan-C), 59.11 (chitosan-
C), 56.65 (chitosan-C), 54.62 (chitosan-C), 30.48 (Ar-O-CH CH ), 29.99
(Ar-O-CH CH CH ), 23.86 (COCH CH ), 22.39 ( P-
), 22.78 (+P-CH
CH CH ) and 18.51 (CH P NMR (202 MHz, CD COOD/D O):
32.52 ppm (singlet, Ph
Poly-N-((3-(3-hydroxy-4-(1-iminoethyl) phenoxy)propyl)triphe-
nylphosphonium hexafluoro- phosphate) oligochitosan (PQPOC ):
Yellow powder, Yield (1.76 g). ATR-FTIR (cm ): 3420 (m, br, ν(O-H +
NH2)), 3247 (m, sh, ν N-H)), 3056 (m, br, ν(Ar-H)), 2962 (m, br, ν(C-H)),
1639 (vs, sh, ν(C=O + C=N)), 1592 (vs, sh, ν(amide II)), 1537 (m, sh), 1439
(s, sh, ν(Ar-P)), 1385 (s, sh, ν(amide III)), 1279 (m, sh, ν(Ar O)), 1149 (s, sh),
1074 (s, sh), 891 (s, sh ν(C-O-C), β-glycosidic linkage), 832 (vs, sh,
(PF6+)), 738 (s, sh). 1H NMR (600 MHz, 1% CD
COOD/D
ppm): 10.28 (s, 2H, 2 x O-H), 8.11 (s, 1H, N-H), 7.68 (d, J =7.0 Hz,
2H, 2 x Ar-H), 7.68 (d, J =7.1 Hz, 12H, 12 x Ar-H), 7.61-7.40 (m, 12H,
2 x Ar-H), 7.37-7.19 (m, 12H, 12 x Ar-H), 6.86 (d, J =6.9 Hz, 2H, 2 x
Ar-H), 6.59 (s, 2H, 2 x Ar-H), 5.61 (s, br, 4H, chitosan-H), 4.28 (t, J
1.4 Hz, 4H, 2 x O-CH ), 4.20-3.98 (m, 12H, chitosan-H), 3.82 (s, br,
2
), 65.13 (chitosan-C), 63.05 (chitosan-C), 62.91
Piroyan,
&
Torchilin, 2012; Boddapati et al., 2005; Yamada
&
2
2
+
Harashima, 2008). As a result, TPP can offer promising motifs for fine-
tuning of the structural features of different polymers to achieve drug
targeted delivery (Wang, Shao et al., 2014, 2014b; Zhang et al., 2015;
Wang et al., 2013; Zhou et al., 2013).
Inspired by the aforementioned amazing facts, the present study is
aimed mainly to in-situ prepare and stabilize AgNPs using new PQP-
based oligochitosans (PQPOCs), safe smart materials, which act as sy-
nergistic reductant and encapsulating agents and thus tightly adhere
AgNPs to fabricate novel nanobiocomposites (NBCs) for antiviral ap-
plications.
2
2
2
3
2
2
31
2
2
3
).
P ).
3
2
+
3
2
−
1
(
-
ν
(
3
2
O)60 ºC δ
2
. Materials and methods
1
2
.1. Experimental protocol
=
2
Extraction of CS from marine wastes and its partial de-polymeriza-
4H, chitosan-H), 3.77 (t, J =6.9 Hz, 4H, chitosan-H), 3.65 (s, br, 4H,
chitosan-H), 3.56-3.36 (m, 8H, chitosan-H), 3.09 (t, J =6.8 Hz, 4H,
tion into oligochitosan (OC) were carried out according to our earlier
work (Elshaarawy et al., 2017). Materials and experimental methods for
preparation of TPP salts (2a,b) along with the different techniques for
comprehensive characterization of all synthesized material were de-
scribed in the electronic supplementary material (ESM).
chitosan-H), 2.53 (t, J =1.3 Hz, 4H, 2 x +P-CH
CH
), 1.94-1.85 (m, 4H, 2 x +P-CH
3H, NHAc), 1.81 (s, 6H, 2 x CH ), 1.33-1.17 (m, 4H, 2 x O-
). C NMR (151 MHz, 1% CD COOD/D O)60ºC δ (ppm):
74.99, 165.12, 163.73, 161.85, 136.43, 132.21, 131.04, 130.01,
2
2
), 2.21- 2.06 (m,
4H, 2 x O-CH
2 2
CH CH
2
2
CH CH ), 1.83 (s,
2
2
3
13
CH
2
CH
2
CH
2
3
2
1
2

A.R. Sofy, et al.
Carbohydrate Polymers 226 (2019) 115261
1
8
6
3
3
18.68, 117.31, 112.24, 109.58, 109.52, 108.39, 105.23, 87.75, 82.81,
2.77, 81.85, 81.63, 80.23, 79.59, 78.93, 75.87, 75.08, 71.83, 71.45,
8.93, 65.13, 63.05, 62.91, 62.88, 61.82, 59.17, 57.00, 55.12, 30.33,
solution (2 mg/mL) was added to each well, and the plates were in-
cubated at 37 °C for 90 min. Then, DMSO (25 μL) was added to each
well to get rid of crystallized formazan. The plates were kept for 15 min
on a shaker to be ready for determination of the optical density at
492 nm (OD492).
3
1
0.02, 23.85, 22.75, 22.61 and 18.48. P NMR (202 MHz, DMSO-d
2.52 ppm (singlet); –142.96 ppm (septet, JPF =711.23 Hz). F NMR
COOD/D O): –70.59 ppm (doublet, JFP =715.68 Hz).
6
):
2
19
1
(
470 MHz, CD
3
2
2.2.3. Antiviral assessment
2
.1.2. In-situ green synthesis of PQPOCs-capped AgNPs
.2 g of PQPOCs was dissolved in 100 mL of Milli-Q water con-
taining 200 μL of NaOH solution (0.3 M) under stirring at 93–95 °C for
h (to ensure maximum deprotonation of the phenol group). After
complete dissolution, the solution was cooled to room temperature and
then a freshly prepared AgNO solution (0.1 M. 3 mL) was added
1 2
The antiviral activity of PQPOCs (PQPOC & PQPOC ) and NBCs
0
(NBC1 & NBC2) have been tested at the concentration (100 μL/mL),
while, the novel most potent biocomposite (NBC1) has been tested at
different serial concentrations (50, 100, 150, and 200 μL/mL) using
Tissue Culture Infective Dose (TCID50) protocol. In brief, solution of
each compound (100 μL) was added to the viral suspension in PBS
2
3
3
dropwise to a PQPOCs solution kept under continuous stirring for a
further 30 min at the same temperature. The color of the solution was
changed from light yellow to orange-yellow. Thereafter, the tempera-
ture of the reaction mixture was raised to 75 °C and stirred at this
temperature for a further 60 min. The solution color was changed from
orange-yellow to yellowish-brown verifying the growth of AgNPs. Then,
the solution was centrifuged and washed five times with Milli-Q water
and absolute ethanol, as well. The final products were dried under
(200 μL, titer ca. 10 TCID50/mL) at pH 7.4. Afterward, the mixture was
vigorously stirred for 10 s and left for 60 min at room temperature to
allow NBC↔virus mutual interaction. Thereafter, the NBC particles
were removed by centrifugation for 10 min at 6000 rpm. The super-
natant (50 μL) was serially diluted several times with PBS in a 96-well
cell culture plate (Greiner-Bio one) containing the appropriate cells
5
(2 × 10 ) to the target virus. Plates were incubated for one hour at
2
37 °C and 5% CO to permit the viral infection. Then, a mixture of
vacuum at 50 °C for 12 h and coded as PQPOC
PQPOC -AgNPs (NBC2).
1
-AgNPs (NBC1) and
(DMEM (50 μL), trypsin (5 ppm) and BSA (0.4%)) was added to each
well once infection occurred, to maintain cells, and allowed for a futher
post-infection for five days. After 5-days post-infection, methanol was
added to fix the surviving cells which stained using Giemsa stain so-
lution (5%). From the number of infected wells, the TCID50 of each
solution was calculated according to Reed-Muench method (Reed &
Muench, 1938). Each compound's antiviral activity is estimated from
the ratio of TCID50 of the NBC-treated supernatant to the viral control
(without treatment). The infectious titers of FCV, HAV, and CoxB4 were
2
2
2
.2. Antiviral study
.2.1. Viruses and cell lines
Cytopathic strain of Hepatitis A virus (HAV) was propagated in Vero
cells according to Yasumura Kawakita protocol (Yasumura & Kawakita,
963). While Coxsackie B4 virus (CoxB4) was cultivated on Hep-2 cell
lines (BioWhittaker, Walkersville, MD, USA) according to Simões work
Simões, Amoros, & Girre, 1999). On the other hand and due to the
1
7
8
7
around 10 , 10 , and 10 TCID50 /mL, respectively.
(
deficiency of cell culture systems or animal models that can be easily
used to grow Norovirus (NoV), the Feline calicivirus (FCV) can be used
as an alternative for mimicking NoV activity, infection behavior, pro-
pagation and survival conditions (Jimenez & Chiang, 2006; D'Souza
et al., 2006). FCV and NoV share numerous biochemical and structural
features such as their similar genomic structure and sequence (3 open
reading frames). So, FCV is considered as a surrogate virus for NoV in
its infectivity and survival studies. FCV (ATCC® VR-2057™, strain FCV-
2.2.4. pH-dependent antiviral activity study
To determine the activity of the most potent nano-biocomposite
(NBC1) at different pH values, a set of test tubes divided into six groups
(in each group, three tubes dedicated to each pH value) containing 9 mL
sterile distilled water (DW) were prepared. All the tubes were exposed
to UV (254 nm, 100 μw/mL) for 30 min before inoculation. The dis-
tribution of groups was as follows:
Group 1: FCV; treated with NBC1.
2280) was grown in Crandell-Reese feline kidney cells (CRFK) ac-
Group 2: FCV; without treatment (control).
cording to earlier work (Gulati, Allwood, Hedberg, & Goyal, 2001). The
cytopathic effect (CPE) of the three viruses has been detected through
their culturing on their specific cell line according to the guided pro-
tocols. Microscopic examination has been carried out daily for ob-
servation of cell morphologies and cell deformation alterations. More-
over, morphological deformations such as loss of confluence, cell
rounding and shrinking, and cytoplasmic granulation and vacuolization
have been recorded in time intervals.
Group 3: HAV; treated with NBC1.
Group 4: HAV; without treatment (control).
Group 5: CoxB4; treated with NBC1.
Group 6: CoxB4; without treatment (control).
7
8
7
A 25 μL of virus suspension (10 , 10 , and 10 TCID50 per mL for
FCV, HAV and CoxB4, respectively) was added to each set of tubes
using the appropriate techniques and maintained at room temperature
for 60 min. Then, 100 μL/mL of NBC1 suspension was added for each
tube; while, control groups were treated with 100 μL of DW. The pH
values have been adjusted by additions of citric phosphate buffer
(0.1 M) to obtain pH range 3–5 while with sodium phosphate buffer
(0.1 M) to get pH range 6-9. The pH values was adjusted to pH 4, 6, 7, 8
and 9 prior viricidal activity assay for each tested group. The virus
dilutions were stored at room temperature for up to 60 min. Then the
antiviral activity of the NBC1 at each pH value was estimated using the
TCID50 /mL. All experiments were done in triplicate.
2.2.2. Cytotoxicity assay
The maximum non-toxic concentration (MNTC) (maximum con-
centration that has no toxic effect and unable to produce any mor-
phological changes in the tested cells relative to the control cells, ex-
pressed in μL/mL) of the novel nano-biocomposites (NBC1,2) on Vero
cells has been estimated by serial dilutions of (10–300 μL/mL). Briefly,
5
2
× 10 cells/mL of Vero cells were treated with the serial dilutions of
the NBC in microtiter plates and have been incubated at 37 °C in a 5%
CO air humidified atmosphere for a further 72 h. Moreover, plates
2
2.3. Statistical methods
were microscopically examined in order to determine the toxic con-
centration of the composite through its ability to induce cell death. The
The collected data were analyzed using Statistical Package for Social
Science (SPSS) for Windows version 25.0. Normality tests were used to
determine whether a given set of data was normally distributed. The
Shapiro Wilk test was used to check for normal distribution of data
(Snedecor & Cochran, 1980). Moreover, all data were quantitatively
presented. Comparing groups was done using One Way ANOVA for
50% cytotoxic concentration of the NBC was defined based on the cell
viability and their ability to cleave the tetrazolium salt, 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma,
Chem, St. Louis, MO) producing formazan (Mosmann, 1983). Briefly,
supernatants were removed from the wells, and 25 μL of an MTT in PBS
3

A.R. Sofy, et al.
Carbohydrate Polymers 226 (2019) 115261
comparison of quantitative data. The P value < 0.05 was considered
statistically significant.
as synergistically capping and reducing agents was depicted in Scheme
2.
3
.2. Degree of acetylation (DA) and degree of substitution (iminization)
3. Results and discussion
(DS)
3
.1. Chemistry of synthesis protocol
The degree of acetylation (DA) of OC and the degree of substitution
DS) in PQPOCs were calculated using its microanalytical analysis
based upon our earlier work (Elshaarawy et al., 2017). The calculated
values were collected in Table 1. These values are consistent with their
CHN analysis.
(
Chitosan, extracted from the marine crustaceans, was used for fab-
rication of oligochitosan (OC), a key starting material for our work,
through the NaNO -based oxidative depolymerization reaction. The
2
amino groups and consequently the degree of acetylation of OC plays a
pivotal role in its chemical modifications via several possible chemical
strategies such as Schiff base reaction, amide reaction, etc.. This to sy-
nergistically improve the aqueous dispersive ability of OC and endow it
more pharmacophores for enhancing of its use in bio-related applica-
tions. Schiff base condensation has been proved to be a facile and ef-
ficient strategy to modify CS surface (Antony, Arun, Theodore, &
Manickam, 2019). In this context, the as-synthesized 3-(4-acetyl-3-hy-
droxyphenoxy)propyl)triphenylphosphonium salts (2a,b) were em-
ployed to impart surface functionalization of OC, covalently, via Schiff
base condensation for production of smart green reducing and capping
agents (PQPOCs) (Scheme 1) which further applied for in-situ bio-
synthesis of AgNPs. The reducing capacity of PQPOCs for Ag+ in the
synthesis of AgNPs could be attributed to the presence of labile re-
ductive phenolic and alcoholic hydroxyl groups upon the surface of
modified OC. Meanwhile, the active coordination sites such as azo-
methine nitrogen and phenolate oxygen scattered on the surface of
PQPOCs backbone allow the concentration of Ag+ ions to their surface
through complexation reaction.
3.3. FTIR spectral data
−
1
Notice of new stretches 1637 ± 2 and 1276 ± 1 cm attributable
for the vibration the imine (C]N) and aryl-O groups, respectively, in
the FTIR spectra of PQPOCs (Fig. 1) confirms a successful covalent
grafting of triphenylphosphonium salts (2a,b) onto the surface of OC
flakes. Resumption of νNH2 band to contribute in the spectra of PQPOCs,
but, with a feeble intensity as compared with that of OC confirms the
partial Schiff base condensation beween triphenylphosphonium salts
−1
(2a,b) and OC. Two prominent peaks around 1112 and 832 cm
are
characteristic for Ph-P+ and PF
phenylphosphonium terminals.
−
vibrations, respectively, of the tri-
6
1
Infrared spectral data collected from the FTIR spectra for PQPOC -
AgNPs (NBC1) provide preliminary evidence for a successful in-situ
green formation and capping of AgNPs. Furthermore, it gives an insight
1
into the potential functional groups of PQPOC backbone responsible
for the reduction of Ag+ ions and capping of AgNPs. The broadness of
the imprint of the OH stretching vibration along with its positive shift in
The potential mechanism for formation AgNPs mediated by PQPOCs
Scheme 1. The schematic diagram for the preparation route of poly quaternary phosphonium-based oligochitosan (PQPOCs).
4

A.R. Sofy, et al.
Carbohydrate Polymers 226 (2019) 115261
Scheme 2. Proposed mechanism for formation of AgNPs mediated by PQPOCs as synergistically capping and reducing agents.
Table 1
a
DA, DS and EA for proposed MF of building unit of OC and PQPOCs .
Sample
DA (%)
DS (%)
MF of building unit (M, g/ mol)
EA Calcd (Found) (%)
C
H
N
OC
PQPOC
PQPOC
26.31
–
–
‒
(C
(C
(C
8
H
8
H
8
H
13NO
13NO
13NO
5
)
5
)
5
)
0.26(C
0.26(C
0.26(C
6
6
6
H
H
H
11NO
11NO
11NO
4
)
4
)
4
)
0.74(H
0.45(C35
0.47(C35
2
O) (190.10)
41.19 (41.24)
54.90 (54.85)
50.00 (49.86)
7.17 (7.21)
6.48 (6.51)
5.90 (5.95)
7.37 (7.29)
4.24 (4.32)
3.86 (3.84)
1
2
28.55
27.08
H
H
37ClNO
NO
6
P)0.29(H
2
O) (324.03)
O) (355.79)
37
F
6
6
P
2
)
0.27(H
2
a
DA = Degree of acetylation; DS = Degree of substitution; EA = Elemental analysis; MF = Molecular formula; OC = Oligochitosan; PQPOC = poly-(quaternary
phosphonium) Oligochitosan.
Fig. 1. Selected FTIR spectral regions for oligochitosan (OC),
poly quaternary phosphonium chloride oligochitosan
1 1
(PQPOC ) and nano-biocomposite1 (NBC1, PQPOC capped
AgNPs). Stretching vibrations ascribed to: †, carbonyl group
formed due to oxidation of phenolic or glycosidic hydroxyl; ‡,
red-shift and demasking of imine; ¶, AgNPs and ¶¶, aryl-O (For
interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.).
the spectra of NBC1 in comparison to that of parent PQPOC
1
confirming
diminishing in the intensity of Aryl–O stretch in the spectra of NBCs
−1
the pivotal role of phenolic and alcoholic OH groups in reduction of
along with the growth of new peak at ∼1683 cm
which can re-
+
Ag ions and stabilization of AgNPs. Meanwhile, a remarkable
cognized as carbonyl stretching mode confirming the oxidation of
5

A.R. Sofy, et al.
Carbohydrate Polymers 226 (2019) 115261
phenolic or glycosidic hydroxyl group. This conclusion is in good
agreement with the earlier outputs of Wang et al. work (Wang, Shao
et al., 2014, 2014b). Moreover, a red-shift of the azomethine stretching
spectra for different NBCs depending on the reducing and capping
agents (PQPOCs) revealed that the structure of PQPOCs played im-
portant roles in size and shape-controlled synthesis of AgNPs.
-1
vibration (Δν = ca. -10 cm ) is derived from the involvement of azo-
methinic N-atom in the coordination of Ag+ ions and stabilization of
AgNPs. Interestingly, observation of a new sharp band in the spectra of
3.6. Morphological characterization
−
1
NBCs at 1450 ± 1 cm , characteristic for AgNPs, evidence the for-
mation and capping of Ag-NPs by PQPOCs.
The surface microstructure of the as-synthesized NBCs (PQPOC
AgNPs and PQPOC -AgNPs) was investigated using SEM technique
Fig. 3A and D). It is clearly seen that the green-synthesized AgNPs with
certain spherical shape are well dispersed on the surface of surface of
PQPOC and PQPOC confirming their successful preparation. An ad-
1
-
2
(
3.4. NMR spectroscopy
1
2
1H-NMR spectra of PQPOCs were recorded in 1% CD
0 °C and are in good agreement with a successful surface-functionali-
zation of OC with PQP brushes as revealed from the observation of
highly de-shielded (downfield) singlets at the ranges of δ = 10.29-
0.26 and 8.11-8.10 ppm characteristic for the resonance of phenolic
ditional evidence for formation and capping of AgNPs by PQPOCs was
provided from energy dispersive x-ray (EDX) analysis, which reveals the
3
2
COOD/D O at
7
existence of atomic Ag, C, O, N and Cl upon the surface of NBC
1
(
Fig. 3C). On the other hand, there is many elements are observed upon
2
the surface of NBC including atomic Ag, C, N, F, P and O (Fig. 3F).
1
These data are clarifying the successful preparation of NBC
in pure form.
1 2
and NBC
and amide protons, respectively, engaged in an intramolecularly H-
1
3
bonded environment. Meanwhile, C-NMR spectra of PQPOCs offer
As shown in the transmission electron microscopy (TEM) images of
NBCs (Fig. 3B and E), AgNPs were spherical in shape and dispersed
within quite a narrow size distribution, which coincides with the
UV–vis result. The surface of the AgNPs was tightly covered by
PQPOCs, which prevented the aggregation of nanoparticles.
further evidence for successful synthesis protocol as detected from no-
1
3
tice of two sets of C-NMR peaks; (i) A downfield set at (175, ∼165,
163 and range of 161-105 ppm) characteristic for the carbonyl,
∼
iminic, phenolic carbons and carbon skeleton of 3-(4-acetyl-3-hydro-
xyphenoxy)propyl)triphenylphosphonium segments, respectively. (ii)
1
3
An up-field C-NMR set in the range of 87-18 ppm ascribed to the re-
sonances of carbon backbone of OC confirming the retention of its
structural identity even after its surface modification. Finally the pre-
3.7. Cytotoxicity
The cytotoxic effects two nano-biocomposites (NBC1 and NBC2)
sence of hexafluorophosphate anion on the surface of PQPOC
2
was
towards Vero cells was investigated using MTT assay. It noticed that
NBC1 is less cytotoxic than NBC2 as revealed from the maximum
nontoxic concentration (MNTC) values (MNTCNBC1 = 200 μL/mL,
MNTCNBC2 = 220 μL/mL). Thus when Vero cells treated with serial
concentrations of NBC1 (up to 200 μL/mL) and NBC2 (up to 220 μL/
mL), no morphological changes were observed in these cells and con-
sequently no inhibition of their enzyme activity.
authenticated by the observation of a septet centered at -144.19 ppm
3
1
19
and a doublet centered at -70.13 ppm in the P/ F NMR spectra of
PQPOC
2
.
3.5. UV–vis absorption spectra
The UV–vis absorption spectra (Fig. 2) demonstrate a divergence
between the as-synthesized AgNPs obtained using different PQPOCs.
Where the symmetrical surface plasmon resonance (SPR) peak observed
around 400 nm was significantly grown for AgNPs prepared and capped
3
3
.8. Antiviral activity
.8.1. Single dose antiviral performance
The antiviral activity of PQPOCs (PQPOC & PQPOC ) and NBCs
1 2
by PQPOC
oped by PQPOC
1
in NBC
1
when compared with AgNPs obtained and envel-
. Moreover, the growth of a new narrow and
2
in NBC
2
(
NBC1,2) have been evaluated at the concentration of (100 μL/mL). It
was found that the lowest percentages of viral reduction 10.57%, 7.25%
and 16% were observed with PQPOC against virions of FCV, HAV, and
CoxB4, respectively with log reduction 0.74 ± 0.30, 0.58 ± 0.21 and
.12 ± 0.17 TCID50/mL of initial viral titers of the three viruses, re-
spectively (Fig. 4). While the antiviral activity of PQPOC showed the
significant log reduction of 1.48 ± 0.08, 1.35 ± 0.043, and
.90 ± 0.100 log10 TCID50/ml (by the 21.1%, 16.91%, and 41.42%
asymmetrical SPR absorbance peak around 530 nm, in the spectra of
NBCs, provids a strong evidence for the successful formation and cap-
ping of AgNPs by PQPOCs. The difference in the UV–vis absorption
2
1
1
2
reduction percentages against FCV, HAV, and CoxB4, respectively.
On the other hand, NBC2 showed higher antiviral activity than
PQPOCs with 4.43 ± 0.04, 2.56 ± 0.05, and 4.84 ± 0.18 log10
TCID50/mL and the reduction percentages of FCV, HAV, and CoxB4
were 63.38%, 31.96%, and 69.19%, respectively. Notworthy, the
treatment with the NBC1 shows the strongest virucidal activity as the
reduction titers were 5.64 ± 0.22, 4.69 ± 0.15, and 5.88 ± 0.08
log10 TCID50/mL and reduction% of FCV, HAV, and CoxB4 were
80.62%, 58.65%, and 84.04%, respectively.
From our study, the novel prepared NBCs (NBC1,2) exhibited
stronger antiviral activity, in comparison to PQPOCs (PQPOC
1
&
PQPOC ), against all the tested viruses. The enhanced antiviral effi-
2
cacies of NBC's as a result of embedding AgNPs into the matrix of
PQPOCs could be attributed to the ability of AgNPs to strongly interact
with the viral glycoproteins, thereby preventing the viral invasion to
the host cell (Mori et al., 2013). To get more insights regarding the
potential role and mode of antiviral action for AgNPs embedded in
NBCs, the effects of the concentration, morphological and physical
features of AgNPs on the antiviral activity of NBCs will be investigated
Fig. 2. UV–vis spectra of the PQPOCs and NBCs. The powdered samples
(0.005 mg) are dissolved in 5 mL of deionized water and then each solution is
monitored using UV–vis spectroscopy.
6

A.R. Sofy, et al.
Carbohydrate Polymers 226 (2019) 115261
Fig. 3. (B and E) TEM images, (A and D) SEM images, and (C and F) EDX of NBC1 and NBC2, respectively.
in the future work. Our trend of results was in good consistency with
the previous studies which revealed that the incorporation nanoma-
terials into a pharmacological formulation had significantly enhanced
its biocidal activity (Kim et al., 2007; Morones et al., 2005).
3.8.3. Effect of pH upon the virucidal activity of NBC1
Effect of pH on the antiviral activity of NBC1 is depicted in Fig. 6
which showed a highly statistically significant difference of antiviral
efficacies against FCV, HAV, and CoxB4 at different pH values (P ≤
0.001). The obtained results clearly showed that at pH 4, the antiviral
activity of NBC1 has reached the maximum level as revealed from the
sharp diminishing in the titer of the inoculated viruses compared to
other pH values. Contrary, this lower pH exert a very mild effect on the
control samples of where the viruses titers were reduced by,
0.46 ± 0.22, 0.21 ± 0.31 and 0.35 ± 0.28 log10 TCID50/mL for FCV,
HAV, and CoxB4, respectively at pH 4. Also, at pH 6 the antiviral effect
of composite was strong against all tested viruses, with the titers re-
covery of 0.56 ± 0.04, 0.86 ± 0.03, and 0.46 ± 0.03 log10 TCID50/mL
corresponding to 8%, 10.78%, and 6.56% respectively, from the in-
3
.8.2. Effect of NBC concentrations of its antiviral performance
The NBC1 showed a virucidal effect against all tested viruses with
concentration-dependent activities profile. Where its activity in the
reduction of infectious FCV virions at different level of concentrations
(
6
50, 100, and 150 μL/mL) are 3.83 ± 0.056, 5.64 ± 0.22, and
.44 ± 0.17 log10 TCID50/mL corresponding to 54.66%, 80.62%, and
2.05% of the reduction%, respectively (Fig. 5). Also, the antiviral ef-
9
fect of the NBC1 against HAV showed a highly significant reduction by
.37 ± 0.045, 4.69 ± 0.15, and 7.05 ± 0.21 log10 TCID50/mL cor-
responding to 29.62%, 58.62%, and 88.12% reduction%, respectively
Fig. 5). On the other hand, the maximum antiviral activity of NBC1was
detected against CoxB4 with reduction of 4.33 ± 0.09, 5.88 ± 0.08,
0.00 TCID50/mL corresponding to 61.85%, 84.04%, and 100%,
7
8
7
2
itially inoculated titers of FCV, HAV and CoxB4 (10 , 10 and 10
TCID50/mL, respectively), with no effect on control virions. At neutral
pH, NBC1 exhibited good antiviral activity as noticed from the recoded
titers values of FCV, HAV and CoxB4 (1.53 ± 0.02, 2.59 ± 0.02,
0.95 ± 0.04 log10 TCID50/mL corresponding to 21.8%, 32.14%, and
13.56%, respectively), while, no significant reduction was observed for
(
7
±
respectively (Fig. 5).
Fig. 4. Titer reduction of FCV, HAV, and CoxB4 treated with PQPOCs (PQPOC
log10 TCID50/mL.
1
& PQPOC ) and NBCs (NBC1,2) at the concentration (100 μL/mL), as detected by
2
7

A.R. Sofy, et al.
Carbohydrate Polymers 226 (2019) 115261
Fig. 5. Infectious titer reduction of FCV, HAV, and CoxB4 after treatment with different concentrations (50, 100, 150, and 200 μL/mL) of NBC1, as detected by log10
TCID50/mL (where letters (a, b, c, d) to show which bars are significantly different).
including their attachment and penetration (Galdiero et al., 2011).
Moreover, Kochkina, Chirkov and others have proposed that CS may
trigger structural damage to the virus capsids causing inactivation as
well as, blocking viral replication, where it can interact with the viral
capsid negative charge (Kochkina & Chirkov, 2000; Su, Zivanovic, &
D'Souza, 2009). Based upon these facts and other reported studies
(
Elechiguerra et al., 2005; Lara, Garza-Treviño, Ixtepan-Turrent, &
Singh, 2011), we can suggest a preliminary mechanism for the antiviral
action of our new NBCs as the AgNPs may prevent the association be-
tween the virus and the host cell based on the direct association of
AgNPs with viral envelope glycoprotein, thereby inhibiting entry of the
virus into host cells. Meanwhile, the inhibition of the viral propagation
by PQPOC component of the NBC could be due to the interference with
the capacity of tested viruses to infiltrate the target cells. Which may be
attributed to either the direct damage the viral moieties or blocking
viral entry. As the tested viruses (FCV, HAV, and CoxB4) belonged to
the non-enveloped viruses, so they have no lipid coats to be attacked by
polycation brushers on the surface of PQPOCs. Thus, it was assumed
that PQPOC served as a viral interior inhibitor by blocking the inter-
action of targeted viruses with the host. We propose that the negatively-
charged binding sites of tested viruses (portal-like form of C-terminal
segment in FCV (Conley et al., 2019); oxyanion hole in CoxB4 (Baxter
et al., 2006); few negative terminals in HAV (Wang et al., 2017)) were
electrostatically interacted with for positive brushes of TPP fragments
in PQPOC which significantly inactivates FCV and CoxB4 more than
HAV, by blocking their cell entry. The significant enhancement in the
antiviral activity by increasing the concentration of NBC, as revealed
from the experimental results (cf. Fig. 5), provides strong evidence for
the reasonableness of our suggestions. Moreover, the remarkable en-
hancement of the antiviral action of NBC1 at acidic medium (pH 4, see
Section 3.8.3) provide strong evidence for this suggestion. Also, the CS
skeleton in PQPOC can curb the propagation of viruses by different
possible mechanisms (Chirkov, 2002): (a) Promoting ribonuclease ac-
tivity which increases the degradation of viral RNA and consequently
prevents its transcription and translation. (b) The glucosamine residue
of CS may hinder the binding between the major viral coat protein
Fig. 6. FCV, HAV and CoxB4 titers treated with NBC1 at different pH values (4,
, 7, 8, and 9) as detected by log10 TCID50/mL.
6
virions in the NBC1-free tubes (controls) at neutral pH. Contrary,
NBC1 has moderate virucidal activity at slight basic pH 8 as shown from
the titers of FCV, HAV and CoxB4 at this pH which was found to be
3.54 ± 0.06, 5.43 ± 0.07 and 2.56 ± 0.12 log10 TCID50/mL with
recovery% of 50.57%, 67.91%, and 36.57%, respectively, however, the
viral reduction in the controls tubes was found to be 0.96 ± 0.23,
4
0.22 ± 0.04 and 0.54 ± 0.2 TCID50/mL for FCV, HAV, and CoxB ,
respectively at this pH, negligible effect. Meanwhile, stronger basic
medium (pH 9) the antiviral activity of NBC1 was feeble (titers values
of FCV, HAV and CoxB4 for treated samples are 5.31 ± 0.08,
7.51 ± 0.13, and 5.84 ± 0.20 log10 TCID50/mL with recovery% 75.9,
93.87%, and 83.47%, respectively), while for controls are 1.78 ± 0.32,
0.38 ± 0.01 and 0.97 ± 0.11 log10 TCID50/mL, respectively. Thus,
our findings indicated that the antiviral activity of NBC1 was pH-de-
pendent.
The fluctuation of the antiviral efficacies for NBCs by changing pH
can be ascribed several possible mechanisms; (i) The acidic pH en-
hances the aqueous solubility of the AgNPs enveloping agents, PQPOCs,
allowing more free and accessible AgNPs for virus interaction. These
capping agents may prevent or weaken the interaction between virions
and AgNPs due to their spatial restriction (Mori et al., 2013). (ii) The
lower pH may induce conformational changes in the viral membrane
fusion proteins and thereby activates their intrinsic fusion activity. (iii)
Lower pH may also play a crucial in triggering capsid uncoating
(
glycoprotein gp120) and their proper receptors.
4. Conclusion
This study reports the synthesis of poly quaternary phosphonium-
(
Greber, Singh, & Helenius, 1994).
based oligochitosans (PQPOC1,2). PQPOCs were used as synergistic re-
ductants for Ag(I) in preparation of silver nanoparticles (AgNPs) and
capping agent to stabilize them through fabrication of PQPOCs-AgNPs
nano-biocomposites (NBC1,2). The new materials were structurally and
morphologically characterized based upon their spectral data (UV–vis,
3.8.4. Proposed mechanism for antiviral action of NBC1
Thus, the use of AgNPs has been extended to the development of
antiviral treatments that inhibit or even reduce their infection,
8

A.R. Sofy, et al.
Carbohydrate Polymers 226 (2019) 115261
FTIR, NMR), electron microscopic examination (SEM, TEM) and EDX
analysis, as well. The outcomes of these spectroscopic and microscopic
analyses revealed the successful formation PQPOCs, AgNPs and NBCs.
Furthermore, the single-dose (100 μL/mL) antiviral performance of the
PQPOCs and NBCs against common human enteric viruses (FCV, HAV,
and CoxB4) reflects a higher virucidal activity for NBCs as compared
with PQPOCs as revealed from the viral reduction% (Viral reduc-
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effect of concentration and pH on the antiviral effect of the most potent
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maximum antiviral at [NBC1] = 200 μL/mL and pH 4. The enhanced
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(
(
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ii) PQPOC served as a viral interior inhibitor by blocking the interac-
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binding sites of viruses. (iii) CS skeleton in PQPOC could induce ribo-
nuclease to degrade the viral RNA and consequently prevents its tran-
scription and translation. In conclusion, the current work initiates a
promising strategy for preparation TPP-based CS/AgNPs composites
which may lead to future integrated antiviral researches.
77, 58–63. https://doi.org/10.1016/j.fct.2014.12.019.
Galdiero, S., Falanga, A., Vitiello, M., Cantisani, M., Marra, V., & Galdiero, M. (2011).
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Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
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