Synthesis of chitosan derivatives with organoselenium and organosulfur compounds: Characterization, antimicrobial properties and application as biomaterials

In this study, Schiff bases of chitosan (CS) were synthesized using citronellal, citral, and their derivatives containing selenium and sulfur. Organoselenium and organosulfur compounds show attractive biological and pharmaceutical activities, which can be beneficial to CS-based materials.

Contents lists available atScienceDirect

Carbohydrate Polymers journal homepage:www.elsevier.com/locate/carbpol

Synthesis of chitosan derivatives with organoselenium and organosulfur

compounds: Characterization, antimicrobial properties and application as

biomaterials

Matheus S Gulartea, João M Anghinonib, Laura Abenanteb, Guilherme T Vossc,

Renata L de Oliveirac, Rodrigo A Vaucherd, Cristiane Luchesec, Ethel A Wilhelmc,

Eder J Lenardãob,⁎, André R Fajardoa,⁎

a Laboratório de Tecnologia e Desenvolvimento de Compósitos e Materiais Poliméricos (LaCoPol), Universidade Federal de Pelotas (UFPel), Campus Capão do Leão,

96010-900, Pelotas, RS, Brazil

b Laboratório de Síntese Orgânica Limpa (Lasol), Universidade Federal de Pelotas (UFPel), Campus Capão do Leão, 96010-900, Pelotas, RS, Brazil

c Laboratório de Pesquisa em Farmacologia Bioquímica (LaFarBio), Universidade Federal de Pelotas (UFPel), Campus Capão do Leão, 96010-900, Pelotas, RS, Brazil

d Laboratório de Pesquisa em Bioquímica e Biologia Molecular de Micro-organismos (LaPeBBioM), Universidade Federal de Pelotas (UFPel), Campus Capão do Leão,

96010-900, Pelotas, RS, Brazil

A R T I C L E I N F O

Chemical compounds studied in this article:

Chitosan (PubChem CID: 71853)

Citronellal (PubChem CID: 7794)

Citral (PubChem CID: 638011)

Poly(vinyl alcohol) (PubChem CID: 3083375)

Glutaraldehyde (PubChem CID: 3485)

2,4-dinitrochlorobenzene (PubChem CID: 6)

Tris-hydrochloride (PubChem CID: 93573)

N,N,N′,N′-tetramethylbenzidine (PubChem

CID: 9702)

Keywords:

Chitosan

Organoselenium

Organosulfur

Schiff base

Antimicrobial activity

Atopic dermatitis

A B S T R A C T

In this study, Schiff bases of chitosan (CS) were synthesized using citronellal, citral, and their derivatives con-taining selenium and sulfur Organoselenium and organosulfur compounds show attractive biological and pharmaceutical activities, which can be beneficial to CS-based materials From the characterization analyses, it was found that the CS-derivatives containing organoselenium and organosulfur compounds exhibited the highest conversion degrees (23 and 28%) Biological assays were conducted usingfilms prepared by the blending of CS-derivatives and poly(vinyl alcohol) The antimicrobial evaluation indicated that thefilm prepared with the sulfur-containing CS was the most active against the tested pathogens (Escherichia coli, Staphylococcus aureus, and Candida albicans) since it reduced considerably their counts (42.5%, 17.4%, and 18.7%) Finally, in vivo assays revealed that thisfilm attenuates atopic dermatitis-like symptoms in mice by suppressing the increase of mye-loperoxidase (MPO) activity and reactive species (RS) levels induced by 2,4-dinitrochlorobenzene (DNCB) In summary, CS-derivatives containing chalcogens, mainly organosulfur, are potential candidates for biomedical applications such as for the treatment of chronic skin diseases

1 Introduction

In the past few years, the use of natural compounds to development

of functional materials for a wide range of application has attracted

great attention, which can be assigned to the attractive and versatile

properties of such compounds (e.g biocompatibility, biodegradability,

low-toxicity, easy availability, among others) (Brodin, Vallejos, Opedal,

Area, & Chinga-Carrasco, 2017; Dugmore, Clark, Bustamante,

Houghton, & Matharu, 2017; Mika, Csefalvay, & Nemeth, 2018) In

particular, polysaccharides, which are claimed as potential substitutes for oil-derived polymers, have been used to elaborate several kind of materials applicable in different areas (biomedicine, pharmacy, agri-culture, hygiene, oil prospecting, among others) (Ali et al., 2018; Guilherme et al., 2015;Huang et al., 2018;Lessa, Gularte, Garcia, & Fajardo, 2017; Ma, Liu, Dong, Wang, & Hou, 2015) Chitosan is one example of polysaccharide largely used for development of novel ma-terials

Chitosan (CS), a well-known chitin derivative, exhibits interesting

https://doi.org/10.1016/j.carbpol.2019.05.040

Received 20 November 2018; Received in revised form 11 February 2019; Accepted 10 May 2019

⁎Corresponding authors at: Programa de Pós-graduação em Química (PPGQ), Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de Pelotas (UFPel), Campus Capão do Leão, 96010-900, Pelotas, RS, Brazil

E-mail addresses:lenardao@ufpel.edu.br(E.J Lenardão),andre.fajardo@pq.cnpq.br(A.R Fajardo)

Available online 11 May 2019

0144-8617/ © 2019 Elsevier Ltd All rights reserved

T

properties such as biodegradability, biocompatibility, nontoxicity,

an-timicrobial activity, among others (Rinaudo, 2006) Despite these

in-trinsic properties, there has been a growing interest in the chemical

modification of CS in order to improve some features and widen its

applicability (Alves & Mano, 2008;Martins et al., 2015) CS is a linear

polysaccharide composed of repeatedβ-(1–4) linked units of either

2-amino-2-deoxy-β-D-glucopyranose (glucosamine) or

2-acetamido-2-deoxy-β-D-glucopyranose (glucosacetamide), depending on the degree

of N-acetylated units (Rinaudo, 2006) It exhibits two types of reactive

groups bound to the main backbone: free amine groups in the

deace-tylated units, and hydroxyl groups in the C3and C6carbon atoms in

acetylated or deacetylated units (Alves & Mano, 2008) The presence of

these groups leads to the possibility of various chemical modifications,

such as the formation of Schiff bases by the reaction with aldehydes or

ketones (Jin, Wang, & Bai, 2009;Yue et al., 2017), the acylation of the

hydroxy groups by reaction with acyl (Santos et al., 2015), the

bioca-talytic oxidation of the hydroxyl groups to aldehydes and carboxylic

acids (da Silva, Krolicka, van den Broek, Frissen, & Boeriu, 2018) and

the sulfonation of the amino and hydroxyl groups, by the reaction with

ClSO3, (Khan & Siddiqui, 2015) among others (Tharanathan & Kittur,

2003) In particular, the free amine groups allow preparing Schiff bases

by reacting them with aldehydes or ketones (linear or aromatics) (Jin

et al., 2009;Yue et al., 2017) Herein, we investigated the synthesis of

Schiff bases by reacting CS with citronellal, citral, and their derivatives

containing selenium and sulfur in order to obtain CS-derivatives with

enhanced biological properties Previous studies demonstrated that

organoselenium and organosulfur compounds have several attractive

biological and pharmaceutical activities (e.g antibacterial, antifungal,

and antioxidant), which can be associated with the ability of selenium

and sulfur to stabilize free radicals (Bhattacherjee et al., 2017;Vogt

et al., 2018) Moreover, the graft of this kind of compound on CS

backbone was not reported in the literature so far

The efficacy of the CS-derivations in biomedical applications was

tested usingfilms prepared by blending of such derivatives with poly

(vinyl alcohol) (PVA), a synthetic polymer with hydrophilic and

bio-compatible properties (Choo, Ching, Chuah, Julai, & Liou, 2016;

Teodorescu, Bercea, & Morariu, 2018) In addition, PVA shows an

ex-cellentfilm-forming ability, which confers to the final material

desir-able mechanical properties As demonstrated in the literature, materials

formulated by crosslinked CS usually exhibit poor mechanical

proper-ties (e.g lack of flexibility, low mechanical strength, etc.), which

re-stricts their application (Kiuchi, Kai, & Inoue, 2008;Vieira, da Silva, dos

Santos, & Beppu, 2011) Taking into account the development of

ma-terials for wound dressing purposes, a promising strategy to overcome

this limitation is to blend CS and PVA to obtain the combined properties

of both polymers Here, in vitro and in vivo experiments were performed

in order to investigate the antimicrobial activities and potential uses of

these CS-derivatives/PVAfilms for the treatment of atopic

dermatitis-like skin lesions

2 Materials and methods

2.1 Materials

All materials utilized in this work are described in Supporting in-formation

2.2 Synthesis of CS-derivatives

CS-derivatives were synthesized via the preparation of Schiff bases

by the reaction of raw CS (1) with different aldehydes (2a-d) The CS (100 mg) was solubilized in acetic acid aqueous solution (40 mL, 1.0 v/ v-% of acetic acid) into a 100 mL round-bottomedflask and heated up

to 55 °C Consequently, an excess of the aldehyde (2 mmol, 2a-d), previously solubilized in ethanol (2 mL), was added to the reaction mixture, which was kept at 55 °C under magnetic stirring for 3 h (see Scheme 1) The amount of aldehydes was calculated based on the amount of the amino groups presented in the CS structure (0.02 mmol

in 100 mg) The resulting CS-derivatives (3a-d) were recovered after the evaporation of the solvent and exhaustively washed with ethanol to remove the non-reacted starting materials Finally, the products were vacuum-dried at 70 °C Trying to know the yield, the excess of aldehyde was quantified and in every cases almost half has been recovered This suggests that the other half reacted with the CS On average of

200 ± 40 mg of the products3a-3d were obtained

2.3 Characterization of the CS-derivatives(3) The products3a-d were characterized using Fourier transform in-frared (FTIR) spectroscopy, Nuclear Magnetic Resonance (NMR), and energy-dispersive X-ray (EDX) analysis FTIR analyses were performed

in a Shimadzu IR-Affinity-1 (Japan) equipment operating in the spectral region of 4000–400 cm−1 Before the spectra acquisition, the products were ground with spectroscopic grade KBr and pressed into disks Hydrogen (1H) NMR spectra were recorded using a Bruker Avance DPX

400 spectrometer at 400 MHz All spectra were acquired using a mix-ture of deuterated solvents (D2O/acetic acid-d410–20 wt-%) and tet-ramethylsilane (TMS) was used as internal standard EDX spectra were recorded using a Jeol JSM-6610LV Scanning Electron Microscopy (SEM) microscope (USA) equipped with an EDX analyzer Before SEM visualization, the samples were gold-coated by sputtering

2.4 Synthesis offilms based on CS-derivatives

To investigate the potential of the CS-derivatives3 in practical uses, films based on the products 3a-d blended with PVA were synthesized by

a solvent casting method In general lines, each CS-derivative 3 (100 mg) was solubilized in 20 mL of acetic acid solution (1.0 v/v-%) and, then, blended with a PVA solution (150 mg in 30 mL of distilled water) The resulting system was homogenized at room temperature for

1 h Next, 25μL of glutaraldehyde (crosslinker agent) was added dropwise to the polymeric solution, which was gently poured into a Petri dish (polystyrene, round-shape) After the solvent casting (va-cuum-oven, 40 °C for 24 h) the as-preparedfilm was peeled off from the Scheme 1 The synthesis of CS-derivatives (3a-d) via Schiff base preparation *Note: Ph = phenyl (C6H5)

Petri dish, immediately washed with distilled water and then

oven-dried (40 °C for 24 h) Thefilm samples synthesized using the different

CS-derivatives were labeled as CS3a-PVA, CS3b-PVA, CS3c-PVA, and

CS3d-PVA, respectively For comparative purposes, afilm sample was

prepared using the raw CS (1) and PVA (labeled here as CS1-PVA)

2.5 Characterization of thefilms

FTIR analyses were performed as aforementioned SEM images were

recorded using a Jeol JSM-6610LV microscope using an acceleration

voltage of 8–10 kV Prior to the SEM visualization, the surface of the

samples was gold-coated by sputtering

2.6 Swelling and stability experiments

Swelling experiments were performed in order to investigate the

liquid uptake capacity of the as-preparedfilms These experiments were

performed using dry samples previously weighed (w0), which were

placed into vials filled with 50 mL of simulated wound fluid (SWF)

(2 w/v-% of bovine albumin, 0.02 mol/L calcium chloride, 0.4 mol/L

sodium chloride, pH 7.4) This system was kept at 37 °C under mild

stirring (100 rpm) and at pre-determined time intervals, the samples

were withdrawn, blotted carefully with tissue paper to remove the

surface-adhered liquid droplets and, then, reweighed (wt) The swelling

degree at different immersion times was calculated using Eq.(1)(Iqbal

et al., 2015) Each swelling experiment was performed in triplicate

= ⎡

⎣

⎦

w

Using a similar gravimetric approach, the stability of the

CS-deri-vativesfilms in SWF was investigated For this, pre-weighed samples

were placed in sealed tubesfilled with 10 mL of SWF and kept at 37 °C

At desired time intervals (1, 2, and 3 weeks), the samples were

with-drawn, washed with ultrapure water to remove undesired compounds

and dried to a constant weight (oven, 50 °C) The stability of eachfilm

sample was calculated using the Eq.(2)

= ⎡

⎣

⎤

⎦

where w0(mg) is the sample initial weight and w1(mg) is the weight

after soaking Again, this experiment was performed in triplicate

2.7 In vitro antimicrobial activity

The antimicrobial activity of the as-synthesizedfilms was evaluated

using the Pour plate method with minor modifications (Iqbal et al.,

2015) Briefly, overnight cultures of Escherichia coli (ATCC 25922),

Staphylococcus aureus (ATCC 6538) and Candida albicans (ATCC 10231) were diluted to adjust a microbial count of 2 × 108CFU/mL For the experiments,film samples were aseptically sectioned (3 × 3 cm) and distributed in sterile Petri dishes These samples were inoculated with

1 mL of tested bacterial solution Immediately after inoculation, ca

15–20 mL of the sterile standard counting agar (PCA) medium was added to the Petri dishes, which were incubated for 24 h at 37 °C under aerobic conditions Petri dishes without the film samples (inoculum only) were used as the control After the incubation period, microbial colonies were counted using a colony counter equipment and the mean log10CFU/mL was calculated The bacterial reduction to evaluate the

effectiveness of the tested materials was calculated as the difference between the log10CFU of the inoculum and the log10CFU recovered from thefilm-containing samples

2.8 In vivo assays

All in vivo assays performed in this work are described in Supporting information

2.9 Statistical analysis

All the collected data in this study were expressed as means ± standard deviation regarding a minimum of three replicates (n≥ 3) The statistical analysis was performed by using OriginPro®8.5 software For in vivo assays, data were expressed as mean ± standard error medium (S.E.M.) Statistical analysis was performed using one-way ANOVA followed by the Newman-Keuls test when appropriated (GraphPad Prism software) Values of p < 0.05 were considered sta-tistically significant

3 Results and discussion

3.1 Characterization of CS-derivatives(3)

In this contribution, FTIR and1H NMR spectroscopic techniques were used to characterize the CS-derivatives (3a-d) as well as to quantify the percentage of substitution in each case The FTIR spectra of raw CS (1) exhibited the characteristic bands associated with this polysaccharide, as noticed in the literature (Fig 1a) (Lawrie et al.,

2007) In this spectrum, it was observed a broad band centered at

3417 cm−1assigned to OeH and NeH stretching vibrations and bands

at 2920 cm-1, 1664 cm-1, and 1600 cm−1due to C–H stretching, amide I and NeH bending from amine and amide II bonds (Lawrie et al., 2007) Additional bands at 1461 cm-1, 1380 cm-1, 1155 cm-1and 1031 cm-1are associated with the−CH2– bending, −CH3symmetrical deformation, antisymmetric stretching of C–O-C and C–N bonds and skeletal

Fig 1 FTIR spectra of raw Cs and Cs-derivatives (A) Full and (B) zoom-in views of the spectra

vibration of C–O stretching (Lawrie et al., 2007; Lessa, Nunes, &

Fajardo, 2018) Overall, the spectra of CS-derivatives (3a-d) exhibited

the bands proceeding from CS with some discrepancies As noticed, the

bands assigned to OeH and NeH stretching vibrations (3600-3100 cm

-1) are sharpened while bands assigned to NeH bending disappeared

indicating that the amine groups of CS (1) have reacted with the

al-dehydes (2a-d) Moreover, the appearance of shoulder-type bands at

1628 cm-1 and bands at 1562 cm-1 confirms the imine (C]N) bond

formation, suggesting that the aldehydes were covalently bound to the

CS backbone (Fig 1b) These data corroborate other similar studies

dealing with the derivatization of CS via Schiff base preparation (Jin

et al., 2009;Marin, Simionescu, & Barboiu, 2012;Tamer et al., 2016)

Furthermore, the spectra of the CS-derivatives (3a-d) also showed

no-ticeable changes in bands associated with the C–H and = C–H vibration

modes (3000-2800 cm-1stretching, 1480-1350 cm-1bending and

1000-650 cm-1out of plane bending) and the appearing of bands at 1652 cm-1

due to C]C stretching of alkenes In particular, the spectra of

CS-de-rivatives containing the –SePh (3c) and –SPh (3d) groups presented

additional bands at 1542 cm-1associated with the C]C stretching of

aromatic rings and bands at 536 cm-1and 648 cm-1assigned to Se-C and

SeC bonds (Devillanova, Sathyanarayana, & Verani, 1978; Vien,

Cotthup, Fatoley, & Crasselli, 1991)

The1H NMR spectrum of raw CS1 (Fig S1) presents the typical

resonance signals assigned to this polysaccharide and their chemical

shifts are in agreement with previous studies (Heux, Brugnerotto,

Desbrieres, Versali, & Rinaudo, 2000) The1H NMR spectra of the

CS-derivatives (3a-d) (Figs S2–S5) exhibited the resonance signals

ex-pected for the CS backbone with some noticeable modifications In

general lines, the CS-derivatives spectra exhibited new resonance

sig-nals in the chemical shift ranges of 1.0–2.0 ppm (−CH3 groups),

1.5–3.5 (aliphatic CH and CH2 groups), and 5.0–5.5 ppm (vinyl

hy-drogen atom) For the CS-derivatives containing the –SePh (3c) and

–SPh (3d) groups, the NMR spectra exhibited new resonance signals in

the region of 7.0–8.0 ppm due to the hydrogen atoms of the phenyl ring

Moreover, all spectra showed new resonance signals attributed to the

imine hydrogen in the region of 9.0–10.0 ppm confirming the

CS-deri-vatization This inference is corroborated by the FTIR data and other

similar studies from literature (Jin et al., 2009;Marin et al., 2012) The

degree of conversion (DC) of–NH2groups of CS in imine (–N = CH–)

units has been estimated using the equation DC = (AN=CH)/(AH1 x

0.85) x 100, where AN=CHand AH1 are the integrated areas of these

respective resonance signals and 0.85 reflects the degree of

deacetyla-tion of the CS used in this study (Marin et al., 2012) The DC values

calculated for3a, 3b, 3c, and 3d were 11%, 18%, 23% and 28%,

re-spectively Table 1 summarizes a general description of the

experi-mental conditions and the calculated DC values for each CS-derivative

As noticed, the highest conversion values were verified to the

al-dehydes containing the–SePh (2c) and –SPh (2d) groups suggesting a

higher reactivity of such compounds as compared with citronellal (2a)

and citral (2b) This result is agrees with previous data reported by

Marin et al (2012), which demonstrated that citral shows low

re-activity with CS when the reaction is performed in aqueous medium On

the other hand, considering the further use of CS in the development of

biomaterials, it is expected that the modification of the CS structure be

restrict to a degree where its original physicochemical and biological

properties remain preserved (Mourya & Inamdar, 2008)

Finally, SEM/EDS analysis was used to investigate the elemental

composition of the CS-derivatives containing the –SePh and –SPh

groups The EDS spectra recorded to3c and 3d compounds (Fig S6)

revealed the presence of signals associated with the elements that

compose the CS chains (C, O, and N) and signals confirming the

pre-sence of the elements Se or S in derivatives3c and 3d Taking together,

these findings confirm the successful synthesis of the CS-derivatives,

including examples of organoselenium and organosulfur compounds

3.2 Characterization of the CS-derivatives basedfilms The chemical crosslinking of the as-synthesizedfilms with glutar-aldehyde was characterized using FTIR spectroscopy The spectra re-corded to eachfilm sample are depicted inFig 2 Glutaraldehyde is a dialdehyde commonly used as a crosslinking agent due to its ability to react with different functional groups Considering the polymers used through in this study (CS and PVA), glutaraldehyde may react primarily with the amino groups of CS and, subsequently, with the hydroxyl groups of PVA As highlighted in the literature, glutaraldehyde converts the amino groups of CS in imine groups and forms acetal rings with PVA (Hoffmann, Seitz, Mencke, Kokott, & Ziegler, 2009;Lessa et al., 2018) The CS1-PVA spectrum shows the characteristic bands of raw CS and PVA with some discrepancies For instance, the broadband assigned to OeH stretching vibration is shifted to higher wavenumber region sug-gesting the decreasing of intramolecular H-bonds due to the cross-linking process Moreover, the obvious increase of intensity regarding the bands associated with the C–H stretching (2900–2800 cm−1) and

C–H bending (1460-1380 cm−1) is assigned to the presence of aliphatic

CH and CH2 groups proceeding from glutaraldehyde and PVA The shoulder band at 1710 cm−1 is due to residual acetate groups re-maining on PVA, the main component of thefilms, while the band at

1652 cm−1is associated with the imine bonds (C]N) formed between the CS and glutaraldehyde, confirming the crosslinking process (Hoffmann et al., 2009; Lessa et al., 2018) The increase of intensity observed for the band at 1458 cm−1can be associated with the increase

of C–N bonds as a result of the incorporation of glutaraldehyde struc-ture in thefilm matrix Finally, the bands at 1200-900 cm−1region are broadened due to the increase of C–O and CeOeC bonds due to the acetal ring formed from the reaction with glutaraldehyde In light of these results, it can be inferred that the CS1-PVA matrix is a full in-terpenetrating network that results from the crosslinking of both polymers with glutaraldehyde A similar analysis can be done from the recorded FTIR spectra of the formulatedfilms using the CS-derivatives

As compared with the Schiff bases (3a-d) (seeFig 1), these spectra exhibited the bands that confirm the blending of Cs-derivatives with PVA and the crosslinking of such polymers with glutaraldehyde It is worth to note that the bands assigned to the imine bonds formed from the reaction between the remaining amino groups from the CS-deriva-tives and the crosslinker are overlapped by the imine bands proceeding from the starting compounds (3a-d)

SEM images of the synthesizedfilms were utilized to investigate their surface morphology (Fig S7, Supporting information) Overall, all films exhibited homogeneous surfaces with poor roughness and without the presence of agglomerates and pores At high magnification, there is not indicative of phase separation in these blended materials suggesting good compatibility between the PVA and the CS/CS-derivatives This good compatibility is likely due to the crosslinking process, which can often generate materials with stable morphologies (Rumer & McCulloch, 2015) It should be mentioned that the presence of some marks on the surfaces of CS3c-PVA and CS3d-PVAfilms is due to slots in the Petri dishes (polystyrene) utilized as a mold From a macroscopic viewpoint, all thefilm samples are visually similar, all of them with someflexibility and mechanically robust for handling This feature can

be assigned to PVA, which improves the mechanical performance of polysaccharides-based materials that sometimes fail to meet specific requirements due to their poor mechanical properties (Teodorescu

et al., 2018)

3.3 Antimicrobial activity

The interest in materials expressing antimicrobial activity has in-creased in the last years, especially in materials designed for biomedical applications For instance, antimicrobial activity is particularly inter-esting for biomaterials applied in the treatment of chronic skin diseases

or skin wounds, because they preventing contamination and infection

of the treated sites and, also, control the spread of pathogens

(Daeschlein, 2013;Dai, Tanaka, Huang, & Hamblin, 2011) Currently,

different approaches have been utilized to endow biomaterials with

antimicrobial properties (e.g chemical modification, incorporation of

synthetic antimicrobials, metal nanoparticles, and so on) (Bilal,

Rasheed, Iqbal, Hu et al., 2017;Bilal, Rasheed, Iqbal, Lib et al., 2017; K

S.Huang et al., 2016) One of the challenges faced by the researchers is

to overcome inherent cytotoxicity issues due to the modification of

these biomaterials In this sense, some elegant strategies have been

reported in the literature to overcome this eventual drawback (Bilal,

Rasheed, Iqbal, Hu et al., 2017;Iqbal et al., 2015) Overall, the use of natural compounds such as plant-based extracts (or their derivatives) seems to be a promising strategy (Iqbal, Kyazze, Locke, Tron, & Keshavarz, 2015;Iqbal, Kyazze, Locke, Tron, & Keshayarz, 2015) Herein, the antimicrobial activities of the preparedfilms against E coli (Gram-negative bacteria), S aureus (Gram-positive bacteria), and C albicans (fungal pathogen) were examined The quantity of microbe after the incubation was evaluated by CFU counts, and the results are displayed inFig 3

As noticed, thefilm synthesized using raw CS (CS1-PVA) exhibited a neglected effect on the reduction of both bacteria counts (E coli and S aureus) and C albicans counts when compared with the control groups

CS shows a well-known antimicrobial activity, which is linked to its cationic groups that interact electrostatically with negatively charged microbial cell membranes inhibiting the growth of microorganisms and leading to the leakage of intracellular electrolytes (Goy, de Britto, & Assis, 2009;Sahariah & Masson, 2017) Generally, the blending of CS with PVA rendersfilms with antimicrobial activity, since this property

is not observed forfilms prepared only with PVA (Bonilla, Fortunati, Atares, Chiralt, & Kenny, 2014) Here, CS was blended with PVA and, further, this blend was crosslinked (i.e reducing the free amino groups

of CS), which probably reduced the positive charge density within the film matrix and impaired the antimicrobial activity In a similar way, thefilm sample synthesized using compound 3b (CS3b-PVA) did not present a noticeable reduction in the microbial counts as compared with the control Although some studies have demonstrated the anti-microbial activity of citronellal (2b) against some strains of bacteria and fungi, such activity is associated with the use of significant amounts

of this monoterpenoid (Lopez-Romero, Gonzalez-Rios, Borges, & Simoes, 2015) According to the NMR data, the percentage of 2b

Table 1

Experimental molar amounts used in this study and the calculated DC values for each CS-derivative

CS-derivatives Tested aldehyde Aldehyde structure Molar amount of amino

groups (mmol) a

Molar amount of aldehyde (mmol)

Amino groups/aldehyde molar ratio

Degree of conversion (%) b

3b Citral

(2b)

3c Phenylselanyl citronellal

(2c)

a Considering 100 mg of CS (deacetylation degree of 85%)

b Calculated from1H NMR data

Fig 2 FTIR spectra of the CS and CS-derivatives basedfilms

grafted in the CS backbone was restricted to 18%, which can explain the

absence of antimicrobial activity in thisfilm Considering the CS3a-PVA

and CS3c-PVAfilms, these films presented some reduction on the

mi-crobe counts; however the difference compared with the control group

and CS1-PVAfilm was not statistically significant In parallel, Cs3d-PVA

reduced the E coli, S aureus and C albicans counts by 42.5%, 17.4%,

and 18.7% (p < 0.05) This result can be associated with the highest

grafting percentage of 2d in the CS backbone as compared with the

other CS-derivatives As demonstrated in the literature, organoselenium

and organosulfur compounds usually exhibit antimicrobial properties

(Schneider et al., 2011;Victoria et al., 2009,2012) In light of this, it

has been noted that the presence of Se or S atoms in the terpenoids (e.g

citral and citronellal) generally enhances the antimicrobial activities of

such compounds as compared with the natural (i.e unsubstituted) ones

(Victoria et al., 2012) As observed from this experiment, it can be

hypothesized that the films synthesized from the CS-derivatives

con-taining organoselenium and organosulfur compounds (CS3c-PVA and

CS3d-PVA) can be efficient devices to control these tested bacteria and

fungi Furthermore, the grafting of such compounds in the CS backbone

allows prolonging the antimicrobial activity of the synthesized films,

thereby preventing microbial re-colonization and proliferation

Con-sidering these results, thefilms CS3a-PVA, CS3c-PVA, and CS3d-PVA,

were selected for further experiments

3.4 Swelling and stability experiments

One of the most important features of an efficient dressing material

applied in wound healing is the capability of such material to absorb and retain the exudate secreted from the wound (Boateng, Matthews, Stevens, & Eccleston, 2008) Materials with low absorption ability are inefficient to remove the wound exudate, leading to its accumulation at the wound surface, which results in microbial attacks and further complications Additionally, hydrophilic materials are able to keep the local moisture, which is perfect for keeping skin and tissue hydrated (Hoque, Prakash, Paramanandham, Shome, & Haldar, 2017) In this sense, swelling experiments were performed in order to investigate the liquid uptake capacity of the selectedfilm samples (CS3a-PVA, CS3c-PVA, and CS3d-PVA) when exposed to SWF The swelling curves built for eachfilm sample are shown inFig 4a

As observed inFig 4, allfilm samples exhibited fast swelling rate just after their immersion in SWF, which can be assigned to the hy-drophilic nature of PVA and CS-derivatives In particular, the liquid uptake in this kind of blend generally increased thanks to the presence

of PVA, which contains a remarkable amount of hydroxyl groups dis-tributed along their molecules (Kamoun, Chen, Eldin, & Kenawy, 2015) Here, the swelling degree of all samples increased rapidly with in-creasing swelling time and close to 60 min after the beginning of the experiment, a discrepant behavior between thefilm samples was ob-served For CS3a-PVA, the swelling rate slows down and the equili-brium was achieved close to 120 min after immersion in SWF The maximum swelling degree calculated for this sample under these ex-perimental conditions was 700% (i.e this sample is able to absorb a liquid amount 7-folds higher than its dry weight) In contrast, CS3c-PVA and CS3d-PVA achieved the equilibrium earlier (˜80 min after Fig 3 (a) E coli, (b) S aureus and (c) C albicans counts (log10CFU/mL) after incubation with different CS-based films Data are reported as mean ± standard error

of the mean (S.E.M.) (One-way analysis of variance/Newman-Keuls test) **p < 0.05 denotes significant levels when compared with control and CS1-PVA groups

Fig 4 (a) Swelling curves and (b) stability of CS3a-PVA, CS3c-PVA, and CS3d-PVA in SWF (pH 7.4) at 37 °C.

Fig 5 Effect of CS-derivatives based films on DNCB-induced atopic dermatitis-like symptoms in mice (a) Images of skin and ear lesions from the groups of mice taken on the last day of the experiment (day 30) (b) Dermatitis scores evaluated on day 30 Data represent the mean ± S.E.M (one-way ANOVA followed by the Newman-Keuls' test).∗ p < 0.05 compared with the control group, # p < 0.05 compared with the DNCB group

immersion) and the maximum swelling degree of these films were

580% and 610%, respectively After the equilibrium, both swelling

curves exhibited a plateau indicating that the liquid absorption leaves

off The highest liquid uptake capacity demonstrated by CS3a-PVA can

be explained due to its lower substitution degree as compared with

other tested CS3c-PVA and CS3d-PVA As aforementioned, compound

3a presented a DS value of 11%, while 3c and 3d have DS values of

23% and 28% Therefore, the film formulated with 3a has more

hy-drophilic groups (i.e amino groups) available in its matrix to interact

with water molecules Sobahi et al (2014) have reported that the

swelling ability of CS-derivatives decrease as a function of their degree

of substitution Despite these finds, it should be mentioned here that

these CS-derivatives/PVA based films exhibited remarkable swelling

properties as compared with other similar devices claimed as wound

dressing materials (Alves et al., 2016;Singh & Dhiman, 2015;Zheng

et al., 2014)

Another desirable feature that a wound dressing material should

possess is an adequate stability Since swellable materials may degrade,

eventually disintegrate and dissolve, it is important to evaluate its

stability when exposed to the wound exudates for a prolonged period

The stability of the selectedfilms in SWF for different periods of time

was examined using gravimetric analyses The results depicted in

Fig 4b, reveal that the CS3a-PVAfilm has the lowest stability against

degradation as compared with the other film samples Three weeks

after its immersion in SWF, the CS3a-PVA sample had just 21% of its

initial weight On the other hand, at the end of the experiment (i.e after

3 weeks), the samples CS3c-PVA and CS3d-PVA still preservers 49%

and 56% of their initial weights Generally, water absorbing materials

with high swelling ability degrade faster, which may explain the results

observed in this experiment, since CS3a-PVA swell more than the other

twofilm samples in similar conditions (Meyvis, De Smedt, Demeester, &

Hennink, 2000) As an additional comment, the imine bonds that keep

the CS moiety crosslinked could be hydrolyzed to the initial amine and

carbonyl groups in the aqueous medium This process is relatively fast

under acidic conditions; however, under neutral or alkaline pH, imine

hydrolysis is relatively slow (Monteiro & Airoldi, 1999), which explains

the results presented inFig 4b On the other hand, PVA should be a

secondary role in this process, since its synthetic polymer shows

no-ticeable resistance against aqueous solubility (Baker, Walsh, Schwartz,

& Boyan, 2012) In summary, it is possible to suggest that the matrix

formed by CS3c-PVA and CS3d-PVA are less susceptible to this

hydro-lysis process than CS3a-PVA

3.5 In vivo assays

3.5.1 Clinical skin severity scores Atopic dermatitis induces edema, erythema, itching, skin pigmen-tation, thickening, eczematous lesions, and excoriation of the skin (Lipozencic & Wolf, 2007) Here, we evaluated the effects of CS-deri-vatives basedfilms treatment on the severity of skin lesions by ap-pearance and clinical skin severity scores in mice exposed to 2,4-dini-trochlorobenzene (DNCB) (Fig 5a and b) [F4,30= 31.15, p < 0.0001] DNCB-induced skin lesion on the murine model is wildly used to study the pathological mechanism of atopic dermatitis or to evaluate the therapeutic candidates for this disease (Jiang & Sun, 2018;Voss et al.,

2018) One-way ANOVA followed by Newman-Keuls' post-hoc test showed that DNCB significantly increased skin severity scores when compared with the control group (p < 0.0001) (Fig 5b) CS3c-PVA and CS3d-PVA treatments partially reduced the severity of skin lesions in-duced by DNCB (p < 0.001) (Fig 5b) In addition, no significant dif-ference in severity of skin lesions was observed after treatment with CS3a-PVA when compared with DNCB group (p > 0.05) (Fig 5a and b)

3.5.2 Scratching behavior

In atopic dermatitis, cutaneous abnormalities such as dryness may initiate an itching sensation that leads to mechanical injury form scratching, and a recurrent itching-scratching cycle can worsen the disease by increasing the release of pro-inflammatory cytokines and chemokine production (Kabashima, 2013) In this sense, with the ob-jective of evaluating the severity of skin lesions caused by DNCB and to provide scientific evidence for CS-derivatives based films anti-pruritus effects, we evaluated the scratching behavior

Effect of CS-derivatives based films on the scratching behavior is showed in Fig 6a [F4,30= 37.18, p < 0.0001] One-way ANOVA followed by Newman-Keuls' post-hoc test revealed that DNCB-exposed animals increased the scratching time, when compared with the control group (p < 0.0001) CS3a-PVA (p < 0.01) and CS3d-PVA (p < 0.001) treatments were partially effective in reducing scratching time, when compared with DNCB group Additionally, CS3c-PVA treatment did not alter the scratching time when compared with DNCB group (p > 0.05) (Fig 6a) Pruritus is a representative feature of atopic dermatitis and triggers a vicious cycle of barrier dysfunction and skin

inflammation, leading to a decrease in the quality of life Here, we verified that CS3a-PVA and CS3d-PVA exert anti-pruritus effects Since there are limited drugs with low side effects and effective inhibition of

Fig 6 Effect of CS-derivatives based films on (a) DNCB-induced scratching incidence and (b) ear swelling Scratching time and ear swelling were evaluated on day

30 Data represent the mean ± S.E.M (one-way ANOVA followed by the Newman Keuls' test).∗ p < 0.05 compared with the control group, # p < 0.05 compared with the DNCB group

itching, well-controlled clinical studies are warranted to demonstrate

the beneficial effects of CS-derivatives based films on atopic dermatitis

Our results of skin lesions and scratching behavior (Figs 5 and 6)

suggested that the animals exposed to DNCB have developed clinical

signs of atopic dermatitis that begin with scratching behavior followed

by the onset of eczematous skin lesions Importantly, CS3d-PVA

treat-ment was effective in reducing both parameters evaluated, suggesting

that this CS-derivative based film has a potential therapeutic in the

treatment and management of atopic dermatitis

3.5.3 Ear swelling

The edema formation is a characteristic feature of atopic dermatitis

In this sense, the anti-edematogenic and anti-inflammatory potentials of

CS-derivatives basedfilms were investigated.Fig 6b illustrates the

ef-fects of CS-derivatives based films on the ear swelling in mice

[F4,30= 52.02, p < 0.0001] One-way ANOVA followed by

Newman-Keuls' post-hoc test demonstrated that DNCB substantially increased ear

swelling as compared with the control group (p < 0.0001) (Fig 6b)

Treatments with CS3a-PVA, CS3c-PVA and CS3d-PVA (p < 0.0001)

partially reduced the ear swelling induced by DNCB (Fig 6b) Our

re-sults indicate that CS-derivatives basedfilms treatments led to a

re-duction of ear swelling induced by DNCB challenge, reflecting an

in-hibition of the edema and cell infiltration in ears Thus, the current

findings suggest that CS3a-PVA, CS3c-PVA and CS3d-PVA exert

anti-inflammatory and anti-edematogenic actions Indeed, other studies

have highlighted the close linkage of inflammation in the

pathophy-siology of atopic dermatitis (Devos et al., 2018; Heratizadeh, 2016;

Voss et al., 2018) In line with our results, it is imperative to understand

the mechanisms that are associated with the anti-inflammatory and

anti-edematogenic actions of CS-derivatives basedfilms For this

pur-pose, the myeloperoxidase (MPO) activity and reactive species (RS)

levels were evaluated

3.5.4 MPO assay

MPO is a myeloid-lineage restricted enzyme with strong

anti-bacterial properties This enzyme is largely expressed by neutrophils,

during myeloid cell diff ;erentiation, which is located within

azur-ophilic granules (Oren & Taylor, 1995) Elevated MPO levels and

ac-tivity are observed in several diseases and the mechanisms whereby

MPO is thought to contribute to disease pathogenesis include tuning of

adaptive immune responses and/or the induction of vascular

perme-ability (Strzepa, Pritchard, & Dittel, 2017)

Fig 7illustrates the effects of CS-derivatives based films on the ear

and back MPO activities [F4,30= 14.58, p < 0.0001 (ear);

F4,30= 19.16, p < 0.0001 (back)] One-way ANOVA followed by Newman-Keuls' post-hoc test revealed that DNCB significantly in-creased the MPO activity in the mouse ear (p < 0.0001;Fig 7a) and back (p < 0.0001;Fig 7b) when compared with the control group The results presented inFig 7a and b demonstrated that treatment with CS3a-PVA, CS3c-PVA or CS3d-PVA (p < 0.0001) reduced the activity

of MPO (ears and back) to levels similar to those in the control group, respectively

The importance of neutrophils in the pathogenesis of a number of autoimmune diseases and the lack of safe and efficient strategies to specifically target them, makes MPO a potential therapeutic target Since neutrophils are important contributors to autoimmune disease pathogenesis it is not surprising that MPO is generally regarded as pathogenic during autoimmune disease progression Herein, in both tissues, all treatments decreased MPO activity induced by repeated DNCB challenges, indicating a reduction in inflammation and corro-borating with the ear swelling results It is important highlight that treatments were applied in the back of animals, suggesting a systemic action In line with these results, we can suggest that the MPO is an important therapeutic target on atopic dermatitis Here, we verify that the modulation of the MPO activity contributes to the protective effects

of CS-derivatives basedfilms on atopic dermatitis

3.5.5 RS levels

RS is considered as one of the important biomarkers of oxidative damage and act as a secondary messenger that can induce the gen-eration of pro-inflammatory and Th2 cytokines during inflammatory signaling (Dormandy, 1978;Kannan & KJain, 2000) Lastly, to explore the linkage of redox imbalance in the pathophysiology of atopic der-matitis, we analyzed the dorsal skin RS levels in mice

Results depicted inFig 8show the effects of CS-derivatives based films on dorsal skin RS levels in mice [F4,30= 10.01, p < 0.0001] One-way ANOVA followed by Newman-Keuls' post-hoc test revealed that DNCB-exposed animals increased the RS levels when compared with the control group (p < 0.001) CS3d-PVA treatment protected against the increase of RS levels induced by DNCB exposure (p < 0.001) CS3a-PVA and CS3c-PVA had no effect in decreasing the RS levels compared with the DNCB group (p > 0.05)

It is well established that skin cells produce RS Atopic dermatitis can disrupt the redox balance, resulting in the overproduction of RS, and high levels of lipid peroxidation products This process exacerbates the disease state and shifts the response toward a Th2 skewed immune response (Briganti & Picardo, 2003) Here, CS3d-PVA treatment alle-viates the increase on RS levels, indicating that its antioxidant activity

Fig 7 Effect of CS-derivatives based films on (a) ear and (b) back MPO activities in mice Data represent the mean ± S.E.M (one-way ANOVA followed by the Newman-Keuls' test).∗ p < 0.05 compared with the control group, # p < 0.05 compared with the DNCB group

probably also contributes to a protective effect on atopic dermatitis.

Indeed, the antioxidant activity of organosulfur compounds has an

important contribution in their pharmacological actions (da Silva et al.,

2017;Ianiski, Alves, Bassaco, Silveira, & Luchese, 2014)

4 Conclusion

In conclusion, we have described the synthesis and characterization

of new chalcogen-containing CS-derivatives Films were prepared by

blending CS-derivatives with PVA, they characterized in detail and

subjected to antimicrobial and pharmacological assays Ourfinds reveal

that thefilm based on CS-modified with the organosulfur compound

(CS3d-PVA) attenuates atopic dermatitis-like symptoms in mice by

suppressing the increase of MPO activity and RS levels induced by

DNCB In addition, its antimicrobial activity seems to contribute to its

pharmacological effect in atopic dermatitis model The present new

finding emphasizes the potential of CS-modified with an organosulfur

compound as a lead material for the development of new agents for the

treatment of atopic dermatitis, a chronic skin disease

Acknowledgments

The authors are grateful for thefinancial support and scholarships

from the Brazilian agencies CNPq (Grant number 305974/2016-5)

CNPq is also acknowledged for the fellowship to A.R.F., E.J.L., E.A.W

and C.L This study was financed in part by the Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior Brasil (CAPES)

-Finance Code 001

Appendix A Supplementary data

Supplementary material related to this article can be found, in the

online version, at doi:https://doi.org/10.1016/j.carbpol.2019.05.040

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Fig 8 Effect of CS-derivatives based films on RS levels of back of mice Data

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com-pared with the DNCB group