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.
Carbohydrate Polymers 219 (2019) 240–250 Contents lists available at ScienceDirect 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 T 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 Leão, 96010900, Pelotas, RS, Brazil b Laboratório de Síntese Orgânica Limpa (Lasol), Universidade Federal de Pelotas (UFPel), Campus Capão Leão, 96010-900, Pelotas, RS, Brazil c Laboratório de Pesquisa em Farmacologia Bioqmica (LaFarBio), Universidade Federal de Pelotas (UFPel), Campus Capão Leão, 96010-900, Pelotas, RS, Brazil d Laboratório de Pesquisa em Bioqmica e Biologia Molecular de Micro-organismos (LaPeBBioM), Universidade Federal de Pelotas (UFPel), Campus Capão Leão, 96010-900, Pelotas, RS, Brazil A R T I C LE I N FO A B S T R A C T 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) 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 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 using films prepared by the blending of CSderivatives and poly(vinyl alcohol) The antimicrobial evaluation indicated that the film 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 this film attenuates atopic dermatitis-like symptoms in mice by suppressing the increase of myeloperoxidase (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 Keywords: Chitosan Organoselenium Organosulfur Schiff base Antimicrobial activity Atopic dermatitis 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, agriculture, 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 materials Chitosan (CS), a well-known chitin derivative, exhibits interesting Corresponding authors at: Programa de Pús-graduaỗóo em Química (PPGQ), Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de Pelotas (UFPel), Campus Capão Leão, 96010-900, Pelotas, RS, Brazil E-mail addresses: lenardao@ufpel.edu.br (E.J Lenardão), andre.fajardo@pq.cnpq.br (A.R Fajardo) 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 Available online 11 May 2019 0144-8617/ © 2019 Elsevier Ltd All rights reserved Carbohydrate Polymers 219 (2019) 240–250 M.S Gularte, et al Scheme The synthesis of CS-derivatives (3a-d) via Schiff base preparation *Note: Ph = phenyl (C6H5) Materials and methods properties such as biodegradability, biocompatibility, nontoxicity, antimicrobial activity, among others (Rinaudo, 2006) Despite these intrinsic 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 repeated β-(1–4) linked units of either 2amino-2-deoxy-β-D-glucopyranose (glucosamine) or 2-acetamido-2deoxy-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 deacetylated units, and hydroxyl groups in the C3 and C6 carbon 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 biocatalytic 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 using films prepared by blending of such derivatives with poly (vinyl alcohol) (PVA), a synthetic polymer with hydrophilic and biocompatible properties (Choo, Ching, Chuah, Julai, & Liou, 2016; Teodorescu, Bercea, & Morariu, 2018) In addition, PVA shows an excellent film-forming ability, which confers to the final material desirable mechanical properties As demonstrated in the literature, materials formulated by crosslinked CS usually exhibit poor mechanical properties (e.g lack of flexibility, low mechanical strength, etc.), which restricts their application (Kiuchi, Kai, & Inoue, 2008; Vieira, da Silva, dos Santos, & Beppu, 2011) Taking into account the development of materials 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/PVA films for the treatment of atopic dermatitislike skin lesions 2.1 Materials All materials utilized in this work are described in Supporting information 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-bottomed flask 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 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 products 3a-3d were obtained 2.3 Characterization of the CS-derivatives (3) The products 3a-d were characterized using Fourier transform infrared (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 mixture of deuterated solvents (D2O/acetic acid-d4 10–20 wt-%) and tetramethylsilane (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 of films based on CS-derivatives To investigate the potential of the CS-derivatives 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 (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 h Next, 25 μL 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 (vacuum-oven, 40 °C for 24 h) the as-prepared film was peeled off from the 241 Carbohydrate Polymers 219 (2019) 240–250 M.S Gularte, et al Staphylococcus aureus (ATCC 6538) and Candida albicans (ATCC 10231) were diluted to adjust a microbial count of × 108 CFU/mL For the experiments, film samples were aseptically sectioned (3 × cm) and distributed in sterile Petri dishes These samples were inoculated with 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 log10 CFU/mL was calculated The bacterial reduction to evaluate the effectiveness of the tested materials was calculated as the difference between the log10 CFU of the inoculum and the log10 CFU recovered from the film-containing samples Petri dish, immediately washed with distilled water and then ovendried (40 °C for 24 h) The film samples synthesized using the different CS-derivatives were labeled as CS3a-PVA, CS3b-PVA, CS3c-PVA, and CS3d-PVA, respectively For comparative purposes, a film sample was prepared using the raw CS (1) and PVA (labeled here as CS1-PVA) 2.5 Characterization of the films 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-prepared films 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 − w0 ⎤ Swelling (%) = ⎡ t x 100 ⎢ ⎦ ⎣ w0 ⎥ 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 statistically significant (1) Using a similar gravimetric approach, the stability of the CS-derivatives films in SWF was investigated For this, pre-weighed samples were placed in sealed tubes filled with 10 mL of SWF and kept at 37 °C At desired time intervals (1, 2, and weeks), the samples were withdrawn, washed with ultrapure water to remove undesired compounds and dried to a constant weight (oven, 50 °C) The stability of each film sample was calculated using the Eq (2) w Stability (%) = ⎡ ⎤ x 100 ⎢ ⎣ w0 ⎥ ⎦ Results and discussion 3.1 Characterization of CS-derivatives (3) In this contribution, FTIR and 1H 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−1 assigned to OeH and NeH stretching vibrations and bands at 2920 cm-1, 1664 cm-1, and 1600 cm−1 due 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-1 and 1031 cm-1 are associated with the −CH2– bending, −CH3 symmetrical deformation, antisymmetric stretching of C–O-C and C–N bonds and skeletal (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-synthesized films was evaluated using the Pour plate method with minor modifications (Iqbal et al., 2015) Briefly, overnight cultures of Escherichia coli (ATCC 25922), Fig FTIR spectra of raw Cs and Cs-derivatives (A) Full and (B) zoom-in views of the spectra 242 Carbohydrate Polymers 219 (2019) 240–250 M.S Gularte, et al 3.2 Characterization of the CS-derivatives based films 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 cm1 ) are sharpened while bands assigned to NeH bending disappeared indicating that the amine groups of CS (1) have reacted with the aldehydes (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 noticeable changes in bands associated with the C–H and = C–H vibration modes (3000-2800 cm-1 stretching, 1480-1350 cm-1 bending and 1000650 cm-1 out 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-derivatives containing the –SePh (3c) and –SPh (3d) groups presented additional bands at 1542 cm-1 associated with the C]C stretching of aromatic rings and bands at 536 cm-1 and 648 cm-1 assigned to Se-C and SeC bonds (Devillanova, Sathyanarayana, & Verani, 1978; Vien, Cotthup, Fatoley, & Crasselli, 1991) The 1H NMR spectrum of raw CS (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) The 1H NMR spectra of the CSderivatives (3a-d) (Figs S2–S5) exhibited the resonance signals expected for the CS backbone with some noticeable modifications In general lines, the CS-derivatives spectra exhibited new resonance signals 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 hydrogen 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-derivatization 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 –NH2 groups of CS in imine (–N = CH–) units has been estimated using the equation DC = (AN=CH)/(AH1 x 0.85) x 100, where AN=CH and AH1 are the integrated areas of these respective resonance signals and 0.85 reflects the degree of deacetylation of the CS used in this study (Marin et al., 2012) The DC values calculated for 3a, 3b, 3c, and 3d were 11%, 18%, 23% and 28%, respectively Table summarizes a general description of the experimental conditions and the calculated DC values for each CS-derivative As noticed, the highest conversion values were verified to the aldehydes 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 reactivity 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 to 3c 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 presence of the elements Se or S in derivatives 3c and 3d Taking together, these findings confirm the successful synthesis of the CS-derivatives, including examples of organoselenium and organosulfur compounds The chemical crosslinking of the as-synthesized films with glutaraldehyde was characterized using FTIR spectroscopy The spectra recorded to each film sample are depicted in Fig 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 suggesting the decreasing of intramolecular H-bonds due to the crosslinking 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 remaining on PVA, the main component of the films, while the band at 1652 cm−1 is 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−1 can be associated with the increase of C–N bonds as a result of the incorporation of glutaraldehyde structure in the film matrix Finally, the bands at 1200-900 cm−1 region 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 interpenetrating network that results from the crosslinking of both polymers with glutaraldehyde A similar analysis can be done from the recorded FTIR spectra of the formulated films using the CS-derivatives As compared with the Schiff bases (3a-d) (see Fig 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-derivatives and the crosslinker are overlapped by the imine bands proceeding from the starting compounds (3a-d) SEM images of the synthesized films 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-PVA films is due to slots in the Petri dishes (polystyrene) utilized as a mold From a macroscopic viewpoint, all the film samples are visually similar, all of them with some flexibility 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 increased in the last years, especially in materials designed for biomedical applications For instance, antimicrobial activity is particularly interesting for biomaterials applied in the treatment of chronic skin diseases or skin wounds, because they preventing contamination and infection 243 Carbohydrate Polymers 219 (2019) 240–250 M.S Gularte, et al Table Experimental molar amounts used in this study and the calculated DC values for each CS-derivative Molar amount of amino groups (mmol)a Molar amount of aldehyde (mmol) Amino groups/aldehyde molar ratio Degree of conversion (%)b Citronellal (2a) 0.02 0.2 1:10 11 3b Citral (2b) 0.02 0.2 1:10 18 3c Phenylselanyl citronellal (2c) 0.02 0.2 1:10 23 3d Phenylthio citral (2d) 0.02 0.2 1:10 28 CS-derivatives Tested aldehyde 3a a b Aldehyde structure Considering 100 mg of CS (deacetylation degree of 85%) Calculated from 1H NMR data 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 prepared films 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 in Fig As noticed, the film 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 renders films with antimicrobial activity, since this property is not observed for films 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, the film 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 antimicrobial 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 Fig FTIR spectra of the CS and CS-derivatives based films 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, 244 Carbohydrate Polymers 219 (2019) 240–250 M.S Gularte, et al Fig (a) E coli, (b) S aureus and (c) C albicans counts (log10 CFU/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 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 selected film samples (CS3a-PVA, CS3cPVA, and CS3d-PVA) when exposed to SWF The swelling curves built for each film sample are shown in Fig 4a As observed in Fig 4, all film samples exhibited fast swelling rate just after their immersion in SWF, which can be assigned to the hydrophilic 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 distributed along their molecules (Kamoun, Chen, Eldin, & Kenawy, 2015) Here, the swelling degree of all samples increased rapidly with increasing swelling time and close to 60 after the beginning of the experiment, a discrepant behavior between the film samples was observed For CS3a-PVA, the swelling rate slows down and the equilibrium was achieved close to 120 after immersion in SWF The maximum swelling degree calculated for this sample under these experimental 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 after grafted in the CS backbone was restricted to 18%, which can explain the absence of antimicrobial activity in this film Considering the CS3a-PVA and CS3c-PVA films, these films presented some reduction on the microbe counts; however the difference compared with the control group and CS1-PVA film 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 containing 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 Considering these results, the films 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 245 Carbohydrate Polymers 219 (2019) 240–250 M.S Gularte, et al Fig (a) Swelling curves and (b) stability of CS3a-PVA, CS3c-PVA, and CS3d-PVA in SWF (pH 7.4) at 37 °C Fig 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 246 Carbohydrate Polymers 219 (2019) 240–250 M.S Gularte, et al Fig 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 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 hydrophilic 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 selected films in SWF for different periods of time was examined using gravimetric analyses The results depicted in Fig 4b, reveal that the CS3a-PVA film 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 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 two film 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 in Fig 4b On the other hand, PVA should be a secondary role in this process, since its synthetic polymer shows noticeable 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 hydrolysis process than CS3a-PVA 3.5 In vivo assays 3.5.1 Clinical skin severity scores Atopic dermatitis induces edema, erythema, itching, skin pigmentation, thickening, eczematous lesions, and excoriation of the skin (Lipozencic & Wolf, 2007) Here, we evaluated the effects of CS-derivatives based films treatment on the severity of skin lesions by appearance and clinical skin severity scores in mice exposed to 2,4-dinitrochlorobenzene (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 induced by DNCB (p < 0.001) (Fig 5b) In addition, no significant difference 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 objective 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 247 Carbohydrate Polymers 219 (2019) 240–250 M.S Gularte, et al F4,30 = 19.16, p < 0.0001 (back)] One-way ANOVA followed by Newman-Keuls' post-hoc test revealed that DNCB significantly increased 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 in Fig 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 corroborating 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 based films on atopic dermatitis 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 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 treatment 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 based films were investigated Fig 6b illustrates the effects of CS-derivatives based films on the ear swelling in mice [F4,30 = 52.02, p < 0.0001] One-way ANOVA followed by NewmanKeuls' 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 results indicate that CS-derivatives based films treatments led to a reduction of ear swelling induced by DNCB challenge, reflecting an inhibition of the edema and cell infiltration in ears Thus, the current findings suggest that CS3a-PVA, CS3c-PVA and CS3d-PVA exert antiinflammatory and anti-edematogenic actions Indeed, other studies have highlighted the close linkage of inflammation in the pathophysiology 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 based films For this purpose, the myeloperoxidase (MPO) activity and reactive species (RS) levels were evaluated 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 generation 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 dermatitis, we analyzed the dorsal skin RS levels in mice Results depicted in Fig show 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 alleviates the increase on RS levels, indicating that its antioxidant activity 3.5.4 MPO assay MPO is a myeloid-lineage restricted enzyme with strong antibacterial properties This enzyme is largely expressed by neutrophils, during myeloid cell diff ;erentiation, which is located within azurophilic granules (Oren & Taylor, 1995) Elevated MPO levels and activity 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 permeability (Strzepa, Pritchard, & Dittel, 2017) Fig illustrates the effects of CS-derivatives based films on the ear and back MPO activities [F4,30 = 14.58, p < 0.0001 (ear); Fig 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 248 Carbohydrate Polymers 219 (2019) 240–250 M.S Gularte, et al Alves, N O., da Silva, G T., Weber, D M., Luchese, C., Wilhelm, E A., & Fajardo, A R (2016) Chitosan/poly(vinyl alcohol)/bovine bone powder biocomposites: A potential biomaterial for the treatment of atopic dermatitis-like skin lesions Carbohydrate Polymers, 148, 115–124 Baker, M I., Walsh, S P., Schwartz, Z., & Boyan, B D (2012) A review of polyvinyl alcohol and its uses in cartilage and orthopedic applications Journal of Biomedical Materials Research Part B-Applied Biomaterials, 100B, 1451–1457 Bhattacherjee, D., Basu, C., Bhardwaj, Q., Mal, S., Sahu, S., Sur, R., et al (2017) Design, synthesis and anti-cancer activities of benzyl analogues of garlic-derived diallyl disulfide (DADS) and the corresponding diselenides ChemistrySelect, 2, 7399–7406 Bilal, M., Rasheed, T., Iqbal, H M N., Hu, H B., Wang, W., & Zhang, X H (2017) Macromolecular agents with antimicrobial potentialities: A drive to combat antimicrobial resistance International Journal of Biological Macromolecules, 103, 554–574 Bilal, M., Rasheed, T., Iqbal, H M N., Lib, C L., Hu, H B., & Zhang, X H (2017) Development of silver nanoparticles loaded chitosan-alginate constructs with biomedical potentialities International Journal of Biological Macromolecules, 105, 393–400 Boateng, J S., Matthews, K H., Stevens, H N E., & Eccleston, G M (2008) Wound healing dressings and drug delivery systems: A review Journal of Pharmaceutical Sciences, 97, 2892–2923 Bonilla, J., Fortunati, E., Atares, L., Chiralt, A., & Kenny, J M (2014) Physical, structural and antimicrobial properties of poly vinyl alcohol-chitosan biodegradable films Food Hydrocolloids, 35, 463–470 Briganti, S., & Picardo, M (2003) Antioxidant activity, lipid peroxidation and skin diseases What’s new Journal of the European Academy of Dermatology and Venereology, 17, 663–669 Brodin, M., Vallejos, M., Opedal, M T., Area, M C., & Chinga-Carrasco, G (2017) Lignocellulosics as sustainable resources for production of bioplastics - A review Journal of Cleaner Production, 162, 646–664 Choo, K., Ching, Y C., Chuah, C H., Julai, S., & Liou, N S (2016) Preparation and characterization of polyvinyl alcohol-chitosan composite films reinforced with cellulose nanofiber Materials, 9, 1–6 da Silva, F D., Pinz, M., de Oliveira, R L., Rodrigues, K C., Ianiski, F R., Bassaco, M M., et al (2017) Organosulfur compound protects against memory decline induced by scopolamine through modulation of oxidative stress and Na+/K+ ATPase activity in mice Metabolic Brain Disease, 32, 1819–1828 da Silva, S B., Krolicka, M., van den Broek, L A M., Frissen, A E., & Boeriu, C G (2018) Water-soluble chitosan derivatives and pH-responsive hydrogels by selective C-6 oxidation mediated by TEMPO-laccase redox system Carbohydrate Polymers, 186, 299–309 Daeschlein, G (2013) Antimicrobial and antiseptic strategies in wound management International Wound Journal, 10, 9–14 Dai, T H., Tanaka, M., Huang, Y Y., & Hamblin, M R (2011) Chitosan preparations for wounds and burns: Antimicrobial and wound-healing effects Expert Review of AntiInfective Therapy, 9, 857–879 Devillanova, F A., Sathyanarayana, D N., & Verani, G (1978) Infrared spectroscopic investigation on N-methyl-1,3-thiazolidine-2-thione and N-methyl-1,3-thiazolidine-2selone Journal of Heterocyclic Chemistry, 15, 945–947 Devos, M., Mogilenko, D A., Fleury, S., Gilbert, B., Becquart, C., Quemener, S., et al (2018) Keratinocyte expression of A20/TNFAIP3 controls skin inflammation associated with atopic dermatitis and psoriasis Journal of Investigative Dermatology, 139, 135–145 Dormandy, T L (1978) Free-radical oxidation and antioxidants Lancet, 1(8065), 647–650 Dugmore, T I J., Clark, J H., Bustamante, J., Houghton, J A., & Matharu, A S (2017) Valorisation of biowastes for the production of green materials using chemical methods Topics in Current Chemistry, 375, 1–8 Goy, R C., de Britto, D., & Assis, O B G (2009) A review of the antimicrobial activity of chitosan Polimeros-Ciencia E Tecnologia, 19, 241–247 Guilherme, M R., Aouada, F A., Fajardo, A R., Martins, A F., Paulino, A T., Davi, M F T., et al (2015) Superabsorbent hydrogels based on polysaccharides for application in agriculture as soil conditioner and nutrient carrier: A review European Polymer Journal, 72, 365–385 Heratizadeh, A (2016) Atopic dermatitis: New evidence on the role of allergic inflammation Current Opinion in Allergy and Clinical Immunology, 16, 458–464 Heux, L., Brugnerotto, J., Desbrieres, J., Versali, M F., & Rinaudo, M (2000) Solid state NMR for determination of degree of acetylation of chitin and chitosan Biomacromolecules, 1, 746–751 Hoffmann, B., Seitz, D., Mencke, A., Kokott, A., & Ziegler, G (2009) Glutaraldehyde and oxidised dextran as crosslinker reagents for chitosan-based scaffolds for cartilage tissue engineering Journal of Materials Science-Materials in Medicine, 20, 1495–1503 Hoque, J., Prakash, R G., Paramanandham, K., Shome, B R., & Haldar, J (2017) Biocompatible injectable hydrogel with potent wound healing and antibacterial properties Molecular Pharmaceutics, 14, 1218–1230 Huang, J J., Deng, Y M., Ren, J A., Chen, G P., Wang, G F., Wang, F., et al (2018) Novel in situ forming hydrogel based on xanthan and chitosan re-gelifying in liquids for local drug delivery Carbohydrate Polymers, 186, 54–63 Huang, K S., Yang, C H., Huang, S L., Chen, C Y., Lu, Y Y., & Lin, Y S (2016) Recent advances in antimicrobial polymers: A mini-review International Journal of Molecular Sciences, 17, 1578–1589 Ianiski, F R., Alves, C B., Bassaco, M M., Silveira, C C., & Luchese, C (2014) Protective effect of ((4-tert-butylcyclohexylidene) methyl) (4-methoxystyryl) sulfide, a novel unsymmetrical divinyl sulfide, on an oxidative stress model induced by sodium nitroprusside in mouse brain: Involvement of glutathione peroxidase activity Journal of Pharmacy and Pharmacology, 66, 1747–1754 Iqbal, H M N., Kyazze, G., Locke, I C., Tron, T., & Keshavarz, T (2015) In situ Fig Effect of CS-derivatives based films on RS levels of back of mice Data represent the mean ± S.E.M (one-way ANOVA followed by the NewmanKeuls' 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) 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 Our finds reveal that the film 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 the financial 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 Coordenaỗóo de Aperfeiỗoamento 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 References Ali, A., Xie, F W., Yu, L., Liu, H S., Meng, L H., Khalid, S., et al (2018) Preparation and characterization of starch-based composite films reinfoced by polysaccharide-based crystals Composites Part B-Engineering, 133, 122–128 Alves, N M., & Mano, J F (2008) Chitosan derivatives obtained by chemical modifications for biomedical and environmental applications International Journal of Biological Macromolecules, 43, 401–414 249 Carbohydrate Polymers 219 (2019) 240–250 M.S Gularte, et al Mourya, V K., & Inamdar, N N (2008) Chitosan-modifications and applications: Opportunities galore Reactive & Functional Polymers, 68, 1013–1051 Oren, A., & Taylor, J M G (1995) The subcellular-localization of defensins and myeloperoxidase in human neutrophils - Immunocytochemical evidence for azurophil granule heterogeneity Journal of Laboratory and Clinical Medicine, 125, 340–347 Rinaudo, M (2006) Chitin and chitosan: Properties and applications Progress in Polymer Science, 31, 603–632 Rumer, J W., & McCulloch, I (2015) Organic photovoltaics: Crosslinking for optimal morphology and stability Materials Today, 18, 425–435 Sahariah, P., & Masson, M (2017) Antimicrobial chitosan and chitosan derivatives: A review of the structure-activity relationship Biomacromolecules, 18, 3846–3868 Santos, J C C., Moreno, P M D., Mansur, A A P., Leiro, V., Mansur, H S., & Pego, A P (2015) Functionalized chitosan derivatives as nonviral vectors: Physicochemical properties of acylated N,N,N-trimethyl chitosan/oligonucleotide nanopolyplexes Soft Matter, 11, 8113–8125 Schneider, T., Baldauf, A., Ba, L A., Jamier, V., Khairan, K., Sarakbi, M B., et al (2011) Selective antimicrobial activity associated with sulfur nanoparticles Journal of Biomedical Nanotechnology, 7(3), 395–405 Singh, B., & Dhiman, A (2015) Designing bio-mimetic moxifloxacin loaded hydrogel wound dressing to improve antioxidant and pharmacology properties Rsc Advances, 5, 44666–44678 Sobahi, T R A., Abdelaal, M Y., & Makki, M S I (2014) Chemical modification of Chitosan for metal ion removal Arabian Journal of Chemistry, 7(5), 741–746 Strzepa, A., Pritchard, K A., & Dittel, B N (2017) Myeloperoxidase: A new player in autoimmunity Cellular Immunology, 317, 1–8 Tamer, T M., Hassan, M A., Omer, A M., Baset, W M A., Hassan, M E., El-Shafeey, M E A., et al (2016) Synthesis, characterization and antimicrobial evaluation of two aromatic chitosan Schiff base derivatives Process Biochemistry, 51, 1721–1730 Teodorescu, M., Bercea, M., & Morariu, S (2018) Biomaterials of poly(vinyl alcohol) and natural polymers Polymer Reviews, 58, 1–40 Tharanathan, R N., & Kittur, F S (2003) Chitin - The undisputed biomolecule of great potential Critical Reviews in Food Science and Nutrition, 43, 61–87 Victoria, F N., Radatz, C S., Sachini, M., Jacob, R G., Alves, D., Savegnago, L., et al (2012) Further analysis of the antimicrobial activity of alpha-phenylseleno citronellal and alpha-phenylseleno citronellol Food Control, 23, 95–99 Victoria, F N., Radatz, C S., Sachini, M., Jacob, R G., Perin, G., da Silva, W P., et al (2009) KF/Al2O3 and PEG-400 as a recyclable medium for the selective alpha-selenation of aldehydes and ketones Preparation of potential antimicrobial agents Tetrahedron Letters, 50, 6761–6763 Vieira, M G A., da Silva, M A., dos Santos, L O., & Beppu, M M (2011) Natural-based plasticizers and biopolymer films: A review European Polymer Journal, 47, 254–263 Vien, D., Cotthup, N., Fatoley, W., & Crasselli, J (1991) The handbook of infrared and Raman characteristic frequencies of organic Molecules Orlando, Florida: Academic Press Vogt, A G., Voss, G T., de Oliveira, R L., Paltian, J J., Duarte, L F B., Alves, D., et al (2018) Organoselenium group is critical for antioxidant activity of 7-chloro-4-phenylselenyl-quinoline Chemico-Biological Interactions, 282, 7–12 Voss, G T., Oliveira, R L., de Souza, J F., Duarte, L F B., Fajardo, A R., Alves, D., et al (2018) Therapeutic and technological potential of 7-chloro-4-phenylselanyl quinoline for the treatment of atopic dermatitis-like skin lesions in mice Materials Science & Engineering C-Materials for Biological Applications, 84, 90–98 Yue, L., Li, J R., Chen, W W., Liu, X L., Jiang, Q X., & Xia, W S (2017) Geraniol grafted chitosan oligosaccharide as a potential antibacterial agent Carbohydrate Polymers, 176, 356–364 Zheng, A., Xue, Y., Wei, D., Li, S., Xiao, H., & Guan, Y (2014) Synthesis and characterization of antimicrobial polyvinyl pyrrolidone hydrogel as wound dressing Soft Materials, 12, 297–305 development of self-defensive antibacterial biomaterials: Phenol-g-keratin-EC based bio-composites with characteristics for biomedical applications Green Chemistry, 17, 3858–3869 Iqbal, H M N., Kyazze, G., Locke, I C., Tron, T., & Keshayarz, T (2015) Development of bio-composites with novel characteristics: Evaluation of phenol-induced antibacterial, biocompatible and biodegradable behaviours Carbohydrate Polymers, 131, 197–207 Jiang, P., & Sun, H (2018) Sulfuretin alleviates atopic dermatitis like-symptons in mice via suppressing Th2 cell activity Immunologic Research, 66, 611–619 Jin, X X., Wang, J T., & Bai, J (2009) Synthesis and antimicrobial activity of the Schiff base from chitosan and citral Carbohydrate Research, 344, 825–829 Kabashima, K (2013) New concept of the pathogenesis of atopic dermatitis: Interplay among the barrier, allergy, and pruritus as a trinity Journal of Dermatological Science, 70, 3–11 Kamoun, E A., Chen, X., Eldin, M S M., & Kenawy, E R S (2015) Crosslinked poly (vinyl alcohol) hydrogels for wound dressing applications: A review of remarkably blended polymers Arabian Journal of Chemistry, 8, 1–14 Kannan, K., & KJain, S (2000) Oxidative stress and apoptosis Pathophysiology, 7, 153–163 Khan, K., & Siddiqui, Z N (2015) An efficient synthesis of tri- and tetrasubstituted imidazoles from benzils using functionalized chitosan as biodegradable solid acid catalyst Industrial & Engineering Chemistry Research, 54, 6611–6618 Kiuchi, H., Kai, W H., & Inoue, Y (2008) Preparation and characterization of poly (ethylene glycol) crosslinked chitosan films Journal of Applied Polymer Science, 107, 3823–3830 Lawrie, G., Keen, I., Drew, B., Chandler-Temple, A., Rintoul, L., Fredericks, P., et al (2007) Interactions between alginate and chitosan biopolymers characterized using FTIR and XPS Biomacromolecules, 8, 2533–2541 Lessa, E F., Gularte, M S., Garcia, E S., & Fajardo, A R (2017) Orange waste: A valuable carbohydrate source for the development of beads with enhanced adsorption properties for cationic dyes Carbohydrate Polymers, 157, 660–668 Lessa, E F., Nunes, M L., & Fajardo, A R (2018) Chitosan/waste coffee-grounds composite: An efficient and eco-friendly adsorbent for removal of pharmaceutical contaminants from water Carbohydrate Polymers, 189, 257–266 Lipozencic, J., & Wolf, R (2007) Atopic dermatitis: An update and review of the literature Dermatologic Clinics, 25, 605–611 Lopez-Romero, J C., Gonzalez-Rios, H., Borges, A., & Simoes, M (2015) Antibacterial effects and mode of action of selected essential oils components against escherichia coli and staphylococcus aureus Evidence-Based Complementary and Alternative Medicine, 2015, 1–9 Ma, Q., Liu, Y F., Dong, Z., Wang, J L., & Hou, X (2015) Hydrophobic and nanoporous chitosan-silica composite aerogels for oil absorption Journal of Applied Polymer Science, 132, 1–7 Marin, L., Simionescu, B., & Barboiu, M (2012) Imino-chitosan biodynamers Chemical Communications, 48, 8778–8780 Martins, A F., Facchi, S P., Monteiro, J P., Nocchi, S R., Silva, C T P., Nakamura, C V., et al (2015) Preparation and cytotoxicity of N,N,N-trimethyl chitosan/alginate beads containing gold nanoparticles International Journal of Biological Macromolecules, 72, 466–471 Meyvis, T K L., De Smedt, S C., Demeester, J., & Hennink, W E (2000) Influence of the degradation mechanism of hydrogels on their elastic and swelling properties during degradation Macromolecules, 33, 4717–4725 Mika, L T., Csefalvay, E., & Nemeth, A (2018) Catalytic conversion of carbohydrates to initial platform chemicals: Chemistry and sustainability Chemical Reviews, 118, 505–613 Monteiro, O A C., & Airoldi, C (1999) Some studies of crosslinking chitosan-glutaraldehyde interaction in a homogeneous system International Journal of Biological Macromolecules, 26, 119–128 250 ... spectra of the formulated films using the CS -derivatives As compared with the Schiff bases (3a-d) (see Fig 1), these spectra exhibited the bands that confirm the blending of Cs -derivatives with PVA and. .. 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. .. Bai, J (2009) Synthesis and antimicrobial activity of the Schiff base from chitosan and citral Carbohydrate Research, 344, 825–829 Kabashima, K (2013) New concept of the pathogenesis of atopic dermatitis: