Supramolecular polyelectrolyte complexes based on cyclodextrin-grafted chitosan and carrageenan for controlled drug release

In the present study, supramolecular polyelectrolyte complexes (SPEC) based on a cyclodextrin-grafted chitosan derivative and carrageenan were prepared and evaluated for controlled drug release. Samples were characterized by FTIR, SEM, and ζ-potential measurements, which confirmed the formation of the polymeric complex.

Carbohydrate Polymers 245 (2020) 116592 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Supramolecular polyelectrolyte complexes based on cyclodextrin-grafted chitosan and carrageenan for controlled drug release T Thamasia F.S Evangelistaa, George R.S Andradeb,*, Keyte N.S Nascimentoa, Samuel B dos Santosc, Maria de Fátima Costa Santosd, Caroline Da Ros Montes D'Ocae, Charles dos S Estevamc, Iara F Gimenezf, Luís E Almeidaa,* a Postgraduate Program in Materials Science and Engineering, Federal University of Sergipe, São Cristóvão, SE, Brazil Postgraduate Program in Energy, Federal University of Espírito Santo, São Mateus, ES, Brazil c Department of Physiology, Federal University of Sergipe, São Cristóvão, SE, Brazil d Posgraduate Program of Chemistry, NMR Laboratory, Departament of Chemistry, Federal University of Paraná, Curitiba, PR, Brazil e NMR Laboratory, Departament of Chemistry, Federal University of Paraná, Curitiba, PR, Brazil f Department of Chemistry, Federal University of Sergipe, São Cristóvão, SE, Brazil b A R T I C LE I N FO A B S T R A C T Keywords: Supramolecular polyelectrolyte complexes Biopolymers Controlled drug release Silver sulfadiazine Silver nanostructures In the present study, supramolecular polyelectrolyte complexes (SPEC) based on a cyclodextrin-grafted chitosan derivative and carrageenan were prepared and evaluated for controlled drug release Samples were characterized by FTIR, SEM, and ζ-potential measurements, which confirmed the formation of the polymeric complex The phenolphthalein test confirmed the presence and availability of inclusion sites from the attached βCD Silver sulfadiazine was used as the model drug and the association with the SPEC was studied by FTIR and computational molecular modeling, using a semi-empirical method DRS and TEM analyses have shown that Ag+ ions from the drug were reduced to form metallic silver nanostructures In vitro tests have shown a clear bacterial activity toward Gram-positive bacteria Staphylococcus aureus and Enterococcus durans/hirae and Gram-negative bacteria Klebsiella pneumoniae and Escherichia coli Finally, this work shows that βCD-chitosan/carrageenan supramolecular polyelectrolyte complexes hold an expressive potential to be applied as a polymer-based system for controlled drug release Introduction The design of local drug release systems (LDRS) based on supramolecular polyelectrolyte complexes (SPECs) has become the subject of fundamental research in the last decade SPECs can be defined as threedimensional macromolecular structures constructed by associating oppositely charged polyelectrolytes in solution (Das & Tsianou, 2017) The SPEC preparation is usually simple, feasible and performed under mild conditions, allowing the final material to have various forms, including nano- and microparticles, membranes, tablets, gels, beads and so on (Luo & Wang, 2014) Also, because of the intrinsic biocompatible nature and the strong and reversible electrostatic interactions between these polycations, the obtained transient structure avoids the use of toxic cross-linkers (Liang et al., 2018), such as epichlorohydrin, allowing them to be used in humans In this context, various natural polymers can be used for preparing SPECs, including chitosan (CS) and carrageenan (CRG) Chitosan is a natural polysaccharide derived from the alkaline Ndeacetylation of chitin, a major structural component of arthropods and crustacean shells Due to its desirable properties, including high biocompatibility, nontoxicity, antifungal, and antimicrobial activities, this cationic biopolymer can be used for a broad range of applications such as food packaging films, drug carriers, wound dressings, polymeric matrix for anchoring metal nanoparticles, and so on (Rezende et al., 2010; Wu et al., 2018) On the other hand, carrageenan is a natural sulfated polysaccharide isolated from red seaweeds (Rhodophyta) and Abbreviations: βCD, β-cyclodextrin; CRG, carrageenan; CS, chitosan; DRS, diffuse reflectance spectroscopy; FTIR-ATR, Fourier transform infrared- attenuated total reflectance; GTM, gentamicin; LDRS, local drug release system; MIC, minimum inhibitory concentration; NMR, nuclear magnetic resonance; PM3, parametric method 3; SPECs, supramolecular polyelectrolyte complexes; SPR, surface plasmon resonance; SSD, silver sulfadiazine; TEM, transmission electron microscopy; v/v %, volume/volume percent; wt %, weight percent; XRD, X-ray diffraction; ZOI, zones of inhibition ⁎ Corresponding authors E-mail addresses: george.andrade@ufes.br (G.R.S Andrade), edu@ufs.br (L.E Almeida) https://doi.org/10.1016/j.carbpol.2020.116592 Received 20 January 2020; Received in revised form 20 May 2020; Accepted June 2020 Available online 11 June 2020 0144-8617/ © 2020 Elsevier Ltd All rights reserved Carbohydrate Polymers 245 (2020) 116592 T.F.S Evangelista, et al its chemical structure consists of a chain of alternating copolymer of βD-galactose and α-D-galactose linked through β-(1, 4) and α-(1, 3) bonds The presence of negatively charged sulfate groups in CRG allows CRG polysaccharide to form a SPEC with CS For instance, various studies have addressed the production and characterization of SPECs based on CS and CRG for various applications, including wastewater purification (Liang et al., 2017), voltammetric glucose biosensor (Rassas et al., 2019), gastroprotective agent (Volod’ko et al., 2014) and controlled drug delivery (Mahdavinia, Karimi, Soltaniniya, & Massoumi, 2019) The use of these SPECs for encapsulating proteins and low molecular weight drugs has raised attention due to practical purposes However, despite the innumerable outstanding advantages, the association of polysaccharide-based SPECs with highly hydrophobic drugs is still a challenge to be overcome In Polymer Science, chemical modifications on biopolymer surface are an easy approach to provide new functionalities for different applications For example, Chen and Wang (Chen & Wang, 2001) reported an easy procedure to modify CS with βcyclodextrin (βCD) for the controlled release of radioactive iodine Cyclodextrins are cyclic oligosaccharides built from 6, 7, and glucose units, respectively α-, β-, and γ-cyclodextrins, that are joined together by α-1,4 bonds Because of the presence and orientation of the hydroxyl groups, these molecules are shaped like a truncated cone, with a hydrophilic outside part and a hydrophobic cavity Thus, the most popular and widely studied property of CDs is the ability to form inclusion complexes with a wide variety of guest molecules based on hydrophobic interactions Host-guest inclusion complexes between βCD and various organic molecules of biological interest were studied via experimental and theoretical approaches (Zhang et al., 2019) For example, Lodagekar and co-workers have shown that the formation of host-guest complexes with cyclodextrins can significantly increase the solubility and dissolution of poorly soluble drugs as well as their pharmacokinetics (Lodagekar et al., 2019) Various papers have shown the ability of cyclodextrin-based polymers to encapsulate small organic molecules for drug delivery applications (El-Zeiny, Abukhadra, Sayed, Osman, & Ahmed, 2020; Ghorpade, Yadav, & Dias, 2017; Kono & Teshirogi, 2015; Tian, Hua, & Liu, 2020) For instance, Campos and co-workers reported the use of β-cyclodextrin-grafted chitosan nanoparticles loaded with volatile organic compounds for designing sustainable biopesticides (Campos et al., 2018) In another work, Hardy and co-workers prepared compact polyelectrolyte complexes based on βCD-functionalized chitosan/alginate for controlled release of anti-inflammatory drugs (Hardy et al., 2018) Herein, different compositions of SPECs based on βCD-grafted chitosan and carrageenan were prepared via electrostatic interactions between the negatively charged −SO3− groups of carrageenan and the positively charged −NH3+ groups of chitosan for local drug delivery system These SPECs, as well as the isolated materials, were fully characterized with FTIR, zeta potential analysis and SEM Also, in order to explore specific applications of the as-prepared SPECs as controlled drug release systems, an inclusion complex with silver sulfadiazine was prepared and characterized Contributions to the understanding of the interactions underlying the formation of the host-guest inclusion complex between the SPEC with silver sulfadiazine were provided by a computational semi-empirical molecular modeling method During the adsorption of the drug, Ag+ ions from the drug were reduced and metallic silver nanostructures were formed, as showed by DRS and TEM analyses To the best of our knowledge, this is the first work reporting the design of SPECs based on βCD-grafted chitosan and carrageenan for controlled release of an antibiotic drug and preparation of silver nanostructures Finally, in vitro studies have shown a clear antibacterial activity toward Gram-positive bacteria Staphylococcus aureus and Enterococcus durans/hirae and Gram-negative bacteria Klebsiella pneumoniae and Escherichia coli Experimental section 2.1 Reagents All the chemicals used in this work were analytical grade and used without further purification: chitosan (CS, Mw =110 kDa, 84 % deacetylation degree, Sigma-Aldrich), β-cyclodextrin (βCD, C42H70O35, Sigma-Aldrich), carrageenan (predominantly κ-carrageenan, CRG, Mw =521 kDa, Sigma-Aldrich), p-toluenesulfonyl chloride (TsCl, C7H7ClO2S, Sigma-Aldrich), phenolphthalein (PhP, C20H14O4, Aldrich), sodium carbonate (Na2CO3, Dinâmica), silver sulfadiazine (SSD, C10H9AgN4O2S, Aldrich), ethanol (C2H6O, Neon), acetic acid (CH3COOH, Vetec), ethoxyethane ((C2H5)2O, Dinâmica), dimethylformamide (DMF, C3H7NO, Neon), Brian Heart Infusion (BHI, Sigma-Aldrich), gentamicin (GTM, C21H43N5O7, Sigma-Aldrich), and pyridine (C5H5N, anhydrous, 99.8 %, Sigma-Aldrich) All aqueous solutions were prepared using Milli-Q ultrapure water (resistivity around 18.2 MΩ cm at 25 °C) 2.2 Synthesis of mono-(6-O-p-toluenesulfonyl)-β-cyclodextrin Prior to grafting βCD with chitosan, a monotosylated βCD derivative (6-OTs-βCD) was prepared by a classic method reported by Matsui and Okimoto (Matsui & Okimoto, 1978) In a typical experiment, solutions of ρ-toluenesulfonyl chloride (30 mL, 6.7 mmol) and β-cyclodextrin (300 mL, 8.8 mmol), both in dry pyridine, were mixed, cooled below °C and stirred overnight Then, the solvent was removed under vacuum at 40 °C using a rotary evaporator (Fisatom 802) and 200 mL of diethyl ether was added to the residue The precipitate was collected, successively recrystallized from water to obtain the pure monotosylated derivative and dried in an oven at 80 °C for 48 h 2.3 Preparation of βCD-CS A method developed by Chen and Wang (Chen & Wang, 2001) was used in this step Initially, 3.0 g of powdered CS was swelled in 150 mL of DMF and 20 mL of a mono-(6-O-p-toluenesulfonyl)-β-CD solution (5.0 g dissolved in DMF) were slowly dropped into the CS solution The mixture was stirred at 140 rpm at 50 °C for 48 h, filtered and washed with water Finally, the powder was dried in an oven at 80 °C for 24 h A yield of 30 % was obtained 2.4 Preparation of βCD-CS:CRG polyelectrolyte complexes SPECs based on βCD-CS (or CS) and CRG were prepared by mixing 10 mL of each polymeric solution in acetic acid (2% v/v) at room temperature, as following: (1) Initially, the polyion stock solutions were prepared at the same concentration (0.181 g L−1) in 40 ml of acetic acid solution; (2) 10 mL of the chitosan gel was added gradually to 10 mL of the carrageenan gel under constant magnetic stirring In this work, different proportions βCD-CS:CRG (or CS:CRG) were studied; (3) After being kept for a given time (24, 48, 72, 96 and 120 h) under magnetically stirring, a residual precipitate mass was collected by centrifugation at 400 rpm for 10 and dried in an oven at 45 °C for 12 h The practical yield was calculated by employing Equation 01: SPEC yield (%) = mSPEC x 100 (mβCD−CS + mCRG ) (1) where mβCD-CS, mCRG, and mSPEC are the initial masses of βCD-CS, CRG, and the final mass of βCD-CS/CRG, respectively 2.5 Evaluation of phenolphthalein inclusion To evaluate if the cyclodextrin’s cavity in βCD-CS is available to form inclusion complexes with organic molecules, an easy test using Carbohydrate Polymers 245 (2020) 116592 T.F.S Evangelista, et al cultures was estimated by comparing the resulting suspension to the McFarland standard For the antimicrobial disc diffusion susceptibility test, an adapted methodology was used (Bauer, Kirby, Sherris, & Turck, 1966) which follows the recommendations from the NCCLS (National Committee of Clinical Laboratory Standards) guidelines (CLSI, 2019) In a typical experiment, cultures of the microorganisms were transferred to mL of BHI agar and incubated overnight at 37 °C The inoculum was standardized and compared to the standard 0.5 tube of McFarland In this work, Petri dishes containing mm of culture medium Muller- Hinton (pH 7.2–7.4) were used Afterward, mm filter discs were impregnated with 20 μg mL−1 corresponding aqueous sample suspension and placed onto the agar surface in each plate containing the microorganisms All the plates were incubated for 12 h at 37 °C As control tests, the bacterial cells without any materials and with gentamicin (20 μg mL−1) were carried out in the same manner The diameters of the zones of inhibition (ZOI) were measured All of these experiments were replicated three times A microdilution method was used for determining the MIC values In a typical experiment, aliquots of 100 μL of the material stock solutions were each diluted in 2-fold serial dilutions in a 96-well plate, with 100 μL of TBS agar and a known amount of the bacteria suspension (108 cfu/mL) Then, the microplates were covered and incubated at 37°C for 24 h The material concentrations varied in half fold according to a standard protocol, ranging from 2000 to 7.81 μg mL−1 in all experiments An analysis of the variation of bactericidal activity over time (a kinetic study) was also performed Initially, a 1.5 × 108 CFU bacterial solution in BHI was prepared and incubated for 18−24 h at 37 °C Then, 0.5 mL of the BHI was transferred to tubes with 4.0 mL of the Müller-Hinton broth: 0.5 mL of the as-prepared material suspension was added to the first; 0.5 mL of sterilized water was added to the second, as negative control; and 0.5 mL of gentamicin was added to the third Then all tubes were intubated in the oven at 37 °C and 100 μL of this mixture were withdrawn after h, h, 12 h and 24 h Finally, the sample was seeded in petri dishes with Müller Hinton and the CFU count was performed on the plate after 18−24 h The results were expressed in Log phenolphthalein was performed The method used here was based on previous works (Mohamed, Wilson, & Headley, 2010; Moreira, Andrade, de Araujo, Kubota, & Gimenez, 2016) and it was chosen because of the strong affinity of phenolphthalein with βCD Initially, a 3.75 mmol L−1 phenolphthalein solution was prepared in ethanol 94 % (94 % ethanol:6 % water, v/v) This solution was diluted with Milli-Q ultrapure water in a 1:10 proportion and the pH was adjusted to 10 by adding a Na2CO3 aqueous solution (1 mol L−1) A known mass of βCDCS was added to 10 mL of the resulting phenolphthalein solution, followed by sonication for h at room temperature (25 °C) Finally, the suspensions were centrifuged at 4000 rpm for10 and the absorbance of the supernatant read at 552 nm Additionally, the same experiment was performed using natural CS, CRG, and βCD-CS:CRG polyelectrolyte complex (4.25CRG:1.0βCD-CS) All the experiments were performed in triplicate 2.6 Complexation of silver sulfadiazine Inclusion complexes based on the as-prepared SPEC with silver sulfadiazine (SSD) were prepared by dispersing 0.6 g of the polymeric matrix (4.25CRG:1.0βCD-CS) in 100 mL of ethanol, then 0.006 g of SSD was slowly added to the reaction mixture and sonicated (40 kHz) for 30 After this step, the mixture was stirred for 24 h The obtained compound was filtered and dried in an oven for 24 h at 45 °C 2.7 Molecular modeling/geometry optimization The molecular modeling methodology was based on previous reports of our group (Borba et al., 2015; de Araújo et al., 2017) Initially, the βCD structure built up based on the data from Cambridge Structural Database (Allen, 2002), whereas the sulfadiazine ion structure was built up using the molecular builder included in Cache Worksystem 6.1 (Fujitsu Ltd., Japan) All the isolated structures were initially optimized employing the MM3 method (Allinger, Yuh, & Lii, 1989, p 3), which is implemented within the Cache Worksystem 6.1 software Then, a complete geometry optimization without an geometric restriction with the Parametric Method (PM3), a semiempirical method implemented within the MOPAC2007 program (Stewart, 1989), was employed The most energetically favorable structures of the isolated molecules (see Fig S1, Supporting Information) were used as starting structures to construct the inclusion complexes The inclusion complex structures were constructed manually by inserting the drug molecule from an end of the host molecule and no geometry constraint was imposed during the optimization Four different inclusion orientations were considered: (1) NH2-in, with the NH2 group pointing toward the narrower βCD rim, (2) NH2-out, having the pyrimidine ring pointing toward the narrower rim, (3) NH2-c-in and (4) NH2-c-out, with the drug molecule positioned in the center of the host cavity All the structures were initially optimized employing the MM3 method and then with PM3 2.9 Characterization The isolated polymers and SPEC samples were characterized by various technics, as described below For SEM-EDS, TEM, NMR, FTIR and UV/visible spectroscopies, the chosen SPEC sample was the one which presented the isoelectric point determined by ζ-potential study (4.25CRG:1.0βCD-CS) 2.10 Net charge determination (ζ-potential) For ζ-potential measurements, a Malvern Zetasizer NanoZS equipment was used This instrument measures the electrophoretic mobility of the sample and calculates the zeta potential using the Smoluchowski expression For this study, various SPEC compositions were prepared by varying the βCD-CS/CRG ratio The samples were prepared as described before (see Section “Preparation of βCD-CS:CRG polyelectrolyte complexes”), where the interaction time was 96 h and pH around 4.5 Then, mL of these samples was placed in a capillary cell (DTS 1070) All measurements were performed at 25 ± °C with the equilibration time set to and without any salt addition or sample dilution 2.8 Antibacterial activity assay Antimicrobial activity of silver sulfadiazine (1.0 %, w/w) loaded βCD-CS/CRG supramolecular polyelectrolyte complex was evaluated using the Mueller-Hinton agar for disk diffusion susceptibility test and by measuring the minimum inhibitory concentration (MIC) values Herein, Gram-positive bacteria Staphylococcus aureus (ATCC 25923) and Enterococcus durans/hirae (SS1225/ IAL 03/10) and Gram-negative bacteria Klebsiella pneumoniae (ATCC 700603) and Escherichia coli (ATCC 25922) were used All the bacterial strains used in this work were donated by the National Institute for Quality Control in Health (INCQS-Fiocruz) For bacteria growth, all apparatus and materials were autoclaved and handled under sterile conditions during the experiments All the bacteria strains were revived with Brain Heart Infusion (BHI) agar at 35°C for h The density of bacterial cells in the liquid 2.11 FTIR spectroscopy FTIR measurements were performed in order to characterize the isolated materials (CS and CRG), the βCD-modified CS and the association between these biopolymers in SPECs FTIR spectra for these powdered materials were recorded on a VARIAN 640-IR FTIR Carbohydrate Polymers 245 (2020) 116592 T.F.S Evangelista, et al Spectrometer using KBr pellets method All the measurements were performed in the wavenumber range 400–4000 cm−1 at a resolution of cm−1 and 64 scans per sample 2.12 UV/visible absorption spectroscopy The UV–vis-NIR absorption spectra of samples in solid-state were obtained by Diffuse Reflectance Spectroscopy (DRS) using an Ocean Optics HR2000 spectrophotometer coupled to an integrating sphere On the other hand, the UV/vis spectra of samples in solution were measured using a Perkin Elmer Lambda 45 spectrophotometer 2.13 SEM-EDS studies The surface morphology of the as-prepared SPECs and the isolated polymers was examined using a JSM-5700 scanning electron microscope (Jeol, Japan) operating at a voltage of kV Elemental analysis of the samples by energy-dispersive X-ray spectroscopy (EDS) was performed at kV using an accoupled EDS detector (Jeol/JCM5700) The surface of the samples was coated with a thin layer of gold by vacuum evaporation The size distribution of the SPEC particles was obtained using the software ImageJ64 by measuring the sizes of more than 100 particles Fig Zeta Potential (mV) for different compositions of βCD-CS/CRG SPECs after 96 h under contact This study was performed by preparing SPECs with different polycation/polyanion mass mixing ratios Initially, the ζ potential was measured for the isolated biopolymers The ζ potential for CS was positive (+92.72 mV), which is related to the positively charged amine groups on its surface After functionalizing this biopolymer with βCD, a decrease was observed in the ζ potential, which reached +48.83 mV This result is expected and confirms the reaction between CS with βCD, which occurs by decreasing the number of free NH2 on the polymer surface The counterpart of the SPEC, CRG, presented a negative ζ potential value (−52.44 mV) due to the existence of negatively charged sulfate groups Fig shows the ζ potential versus different βCD-CS:CRG compositions, differing the mass mixing ratio From an exponential fit applied to these data, it was observed that this SPEC system is characterized by the presence of two important zones The first one goes from point (I) to (II), where the ζ potential became significantly less negative as the ratio βCD-CS/CRG varies from 0.12 (1:8, βCD-CS:CRG) to 0.22 (1:4.5) The point (II) in Fig corresponds to the isoelectric point, where ζ potential is zero Above that point, a new zone (from point (II) to (III)) is characterized by a charge switchover from negative to positive values, indicating the excess of βCD-CS The excess of the polyanion or the polycation counterpart during the preparation can afford the design of cationic or anionic SPECs, which may occur via different mechanisms The formation of cationic SPEC particles can be described by the hydrophilic crown mechanism (Volod’ko et al., 2018) This mechanism is based on the stabilization of the system by unreacted amine groups from the polycation, which are located outside the SPEC particles On the other hand, as observed in Fig 1, anionic SPEC particles are formed when the βCD-CS:CRG ratio is below 0.22 (point II) In this case, the excess of κ-CRG molecules enhances the negative charge density on the SPEC surface 2.14 Transmission Electron Microscopy (TEM) The samples were characterized by TEM using a Jeol JEM-1400 Plus instrument operated at 120 kV Samples were previously diluted in water (1:10, v/v) and then deposited onto copper grids coated with ultrathin carbon and formvar films 2.15 NMR analysis The samples (10 mg) were dissolved in D2O/DCl % v/v solution (600 μL) at room temperature 1H NMR spectra were acquired on an Bruker AVANCE II 400 NMR spectrometer (Bruker BioSpin Corporation) equipped with a mm multinuclear direct detection probe with z-gradient, operating at 9.4 T observing the 1H nuclei at 400.13 MHz at 80 °C All NMR spectra were acquired through pulse sequence zg (Bruker library), 64 K data points, spectral width 14 ppm and 32 or 64 transients The chemical shifts were expressed in relative to methyl group of internal reference, TMSP-d4 at δH = 0.00 Results and discussion 3.1 SPEC yield and net charge determination Herein, SPEC materials based on a βCD-grafted CS and CRG were prepared for controlled drug release When the carrageenan solution is dropped into the chitosan solution, instantaneous turbidity was observed due to the electrostatic attraction between positively charged amine groups on CS and negatively sulfate groups on CRG Depending on the interaction time between the used biopolymers, different mass yields can be achieved as a result of the kinetic formation of the SPEC, which can involve nucleation, growth, and rearrangement steps (Takahashi, Narayanan, & Sato, 2017) The yield of SPEC was calculated based on the complex mass obtained after the drying process and the initial mass of the isolated biopolymers Table S1 (see Supporting Information) shows that the interaction between βCD-CS and CRG produced the highest complex yield around 66.5 % after 96 h under continuous stirring After 120 h, the SPEC yield was very close to the one found at 96 h, which suggests that the complex reached its highest yield This interaction time was used to prepare all the samples in this work Zeta (ζ) potential was used to investigate the charge density of the as-prepared SPECs and to determine the isoelectric point of this system 3.2 Structural and molecular characterization of the isolated biopolymers and SPECs FTIR spectroscopy was used to explain the interaction between the polyelectrolytic biopolymers The functional groups of CS, βCD-CS, CRG, and βCD-CS:CRG supramolecular polyelectrolytic complex were studied using FTIR analysis, as shown in Fig The pure chitosan exhibited the following characteristic peaks: (1) a broadband at 3800 to 2985 cm−1, relative to OH asymmetric vibrations; (2) superimposed to this large band, a contribution at 3360 cm−1 can be attributed to a stretching mode of NH from a group located at the polysaccharide chain’s glucose residue; (3) another peak was found at 2880 and 2930 cm−1, which is relative to CH groups; (4) a characteristic peak from amide I at 1655 cm−1, (5) a band of -NH2 bending at 1590 cm−1; (6) bands at 1420 cm−1 (CH2 deformation), 1383 (-CH3 symmetric Carbohydrate Polymers 245 (2020) 116592 T.F.S Evangelista, et al some peaks are overlapped However, the presence of a characteristic peak at 944 cm−1, assigned to the α-pyranyl vibration of CD, and also the absence of a characteristic peak of the benzene backbone from Ts group (a peak at approximately 1605 cm−1) corroborates the reaction between βCD and CS (Chen & Wang, 2001) The FTIR spectrum of free carrageenan exhibited a broad vibration band from OH moieties at 3450 cm−1, corresponding to OH stretching, and bands around 2910 cm−1, which can be assigned to stretching vibration of hydrocarbon groups (CH3 and CH2) Typical peaks of sulfate ester groups were found between 1210 and 1260 cm−1 (Dong et al., 2018; Rhein-Knudsen, Ale, Ajalloueian, Yu, & Meyer, 2017) A strong band at 930 cm−1 indicated the presence of 3,6-anhydro-D-galactose and a band at 840 cm−1 can be assigned to galactose-4-sulfate (Roy & Rhim, 2019) The polycation and the polyanion macromolecules interact mainly via electrostatic attraction, although inter-macromolecular interactions, including H-bonding, hydrophobic interactions, dipole interactions, and van der Waals forces can also be involved The result of these interactions is the formation of a precipitate, the SPEC In Fig 2, it is possible to find the characteristic bands from amide and sulfate groups in the SPEC, confirming the presence of both biopolymers, although no significant changes have been observed in the band positions (Carneiro et al., 2013) The chemical structures of CS, βCD-CS, SPEC, SSD, and SSD/SPEC were fully characterized by 1H NMR For unmodified CS (see Fig S3a, Supporting Information), signals from anomeric hydrogens (H-1) can be clearly identified at δH 4.94 (d, J =7.9 Hz) and 4.65 (d, J =7.3 Hz) for glucosamine (D) and N-acetylglucosamine (A), respectively Moreover, the signal at δH 2.06 (s) can be attributed to hydrogens from methyl group of N-acetylglucosamine (H3C-Ac) and the signal at δH 3.24 (H2) of glucosamine (D) The multiplet proton signals at δH 4.03−3.55 were attributed to the hydrogens 2−6 All assignment were characteristic of CS and have also been previously reported (Auzély-Velty & Rinaudo, 2001; Lavertu et al., 2003) For determining the degree of acetylation (DA%) of chitosan (CS), we used the integrals of the hydrogens from the methyl group of N-acetylglucosamine (H3C-Ac) and hydrogen (H-2) of glucosamine (D) The formula used was DA (%) = (ACH3/3 AH2) × 100 The analysis showed that commercial CS presents an acetylation degree of 16 % (Fig S3), which is similar to the one described by the supplier (15 %) Fig S3 (see Supporting Information) shows the 1H NMR of βCD-CS The degree of β-CD substitution on chitosan was determined as reported by Venter et al (Venter, Kotzé, Auzély-Velty, & Rinaudo, 2006), using the following equation: Fig FTIR spectra of the isolated components and SPECs deformation), and 1323 cm−1 (amide III and CH2 wagging); and (7) bands related to the glycosidic ring at 1200−800 cm−1 (the glycosidic linkage appears at 1156 cm−1) All these observations are in agreement with previous studies (Liang et al., 2017, 2018) The chemically active groups for CS are the free NH2 and OH groups, both of which prone to be modified and involved in hydrogen, hydrophobic or electrostatic bonds (Mohammed, Syeda, Wasan, & Wasan, 2017) In this work, the CS surface was modified with 6-OTsβCD This monotosylated βCD derivative is widely used as an intermediate for transforming the primary hydroxyl moiety of the βCD into other functional groups As observed in Fig S2 (see Supporting Information), the reaction is processed with the removal of the leaving group (OTs) and the direct attachment of βCD via NH2 group from CS The FTIR spectrum for βCD-CS is shown in Fig βCD and CS are both carbohydrates, so they have some similar groups and, consequently, DS (%) = H1CD x 100 H2'Chitosan (2) where, DS is the degree of substitution, H1CD is the integration value of the H1 anomeric proton of βCD signal and H2’Chitosan is the integration value of the H2’ proton signal Thus, the found value for DS was 18.45 % Also, it was observed a signal related to the tosyl group at δH Fig 1H NMR (D2O/DCl, % v/v, 400 MHz, 80 °C) spectra: (a) CS/βCD (1), CRG (2) and SPEC (3) (b) SPEC (1), SDZ-Ag (2), SSD/SPEC (3) Carbohydrate Polymers 245 (2020) 116592 T.F.S Evangelista, et al Fig SEM images of CS (A and B), β-CD (C and D), βCD-CS (E and F), CRG (G and H) and SPEC (I and J) Carbohydrate Polymers 245 (2020) 116592 T.F.S Evangelista, et al 2.4, which is absent in the 1H NMR spectra of SPEC (see Fig 3) Its narrow area suggests that it is merely a small remaining fraction from the synthesis The interaction between CS/β-CD with carrageenan (CRG) was also studied by 1H NMR spectra (Tojo & Prado, 2003) As observed in Fig 3a, it is clean the change of the proton chemical shifts for both biopolymer, especially for the H2’ proton signal and for the chemical shifts from δH 5.5 to 4.5 According to Voron’ko et al changes like this for polyelectrolyte complexes indicate a change in the electrostatic interactions involving charged groups from the polyions (Voron’ko, Derkach, Vovk, & Tolstoy, 2016) The surface morphology of the isolated materials and the as-prepared SPEC was studied by SEM analysis Fig 4a and b show the surface structure of commercial CS, which exhibited a flake-like structure with a moderate degree of irregularity βCD (Fig 4c and d) presented a three-dimensional block structure with an irregular shape After the functionalization of CS with βCD, the final polymer structure was smoother than the initial CS, as observed in Fig 4e and f Commercial CRG also presented an irregular morphology (see Fig 4g and h) On the other hand, the mixing of aqueous βCD-CS and CRG solutions leads to the formation of a dense phase, which is observed in the SEM images as well-defined SPEC sub-micro particles (Fig 4i and j) The precipitation of the SPEC as sub-micro particles fully confirms the electrostatic interactions between the oppositely charged macromolecules 3.3 Evaluation of phenolphthalein inclusion Fig Evaluation of phenolphthalein inclusion into different biopolymers Curve of [PhP]/[PhP]0 versus [Biopolymers], where [Biopolymers], [PhP]0, and [PhP] are the concentration of the biopolymer, the initial and final concentrations of PhP, respectively Herein, the as-prepared SPECs were designed to act as drug delivery/release systems mainly by forming host-guest inclusion complexes Thus, the first step is to evaluate the availability of inclusion sites, which can be easily determined by absorbance changes of a suitable organic dye when in contact with these materials Phenolphthalein (PhP) is a phthalein dye generally used as a pH indicator because of its distinctive color change from colorless to pinkpurple in pH values above 8.4 When PhP is added to a CD aqueous solution, a decrease of absorbance in the visible region (λmax =552 nm) is observed, even in alkali solutions, due to a host-guest complex formation This behavior was explained by Taguchi (Taguchi, 1986) in terms of the transformation of PhP into its colorless lactonoid dianion form within the host cavity Thus, because of its high affinity for the CD cavity, PhP has been used for proving the presence of inclusion sites and, consequently, the existence of cyclodextrins (Akỗakoca Kumbasar, Akduman, & Çay, 2014) As observed in Fig 5, when the SPEC containing βCD is added to a PhP solution, a significant decrease of absorbance intensity at the absorption maxima (λ =552 nm) occurs, which 95 % of the absorbance decrease for the lowest concentration of SPEC (1.0 mg/mL) This result indicates the successful formation of supramolecular graft polymer structures, as the βCD molecules in the SPEC surface are able to form host-guest complexes with organic molecules However, it is well known that biopolymers and their derivatives are highly active towards adsorption of dye molecules This interaction can occur via physical adsorption (when the drug is physically entrapped) or via a variety of intermolecular attraction forces, which depends on the chemical nature of all the components Thus, a control test consisting of mixing the same mass of CS, CRG and their complex (CS/CRG) with a PhP solution was performed to evaluate the adsorption of the dye in the absence of βCD As observed in Fig 5, the adsorption test demonstrated a small contribution to the decrease of the PhP absorbance at 552 nm As βCD is absent in CS/CRG (as well as in bare CS and CRG), this result suggests that there is interaction between PhP and the polymeric chain Likewise, the same study was performed in the presence of the βCD-grafted CS and the absorbance decrease was around 85 % for the highest concentration of this polymer (6.0 mg/mL) When βCD-CS and SPEC are compared, it is expected a higher decrease of PhP absorbance when the SPEC is used, as PhP is able to interact not only by the formation of inclusion complexes Those tests evidence the presence of βCD in the as- prepared SPEC and the possibility of preparing host-guest complexes 3.4 Evaluation of SSD inclusion Herein, SSD was incorporated into the as-prepared SPEC SSD was chosen as a model drug because it is an effective antibacterial agent with a broad spectrum of activity against various bacterial strains, including P aeruginosa and S aureus SSD is commonly used for topical treatment of burn wounds, as the silver ions act both as a bacteriostatic and as a bactericidal agent The SPEC which presented neutral zeta potential charge and higher stability in water was chosen as the drug carrier vehicle The incorporated amount of SSD in the SPEC was determined using UV–vis spectroscopy, by comparing the initial absorbance intensity at λ = 256 nm (AInitial = 1.7034) and final absorbance after 24 h under contact with the SPEC (AFinal = 0.1790) So, it is observed a decrease of 89.5 % of the absorbance intensity, which can be related to the incorporated amount of the drug in the polymer matrix After adding a known amount of SSD to the SPEC suspension under ultrasonic irradiation, the system color changes from pale yellow to purple In order to investigate this behavior, SSD/SPEC and the isolated samples were characterized by DRS and TEM As observed in Fig 6, the DRS spectrum of SSD/SPEC presents a shoulder at 360 nm related to the guest drug In addition, the SSD/SPEC spectrum presents a broad band centered at 536 nm, which is absent in the bare biopolymers and SSD spectra The presence of this broadband can be related to the conversion of Ag+ ion to metallic Ag° According to Bastús et al., large metallic silver particles, prepared using a mixture of two reducing agents (sodium citrate and tannic acid), present bands around 500 nm, which can be related to the surface plasmon resonance (SPR) (Bastỳs, Merkoỗi, Piella, & Puntes, 2014) SPR for metallic silver particles can be understood as the collective oscillation of electrons in resonance with the frequency of the incident electromagnetic radiation In another work, Jiang and coworkers reported the preparation of anisotropic silver particles using a mix of reducing agents, including citrate and sodium bis(2-ethylhexyl)sulfosuccinate (Jiang, Chen, Chen, Xiong, & Yu, 2011) In their study, the Carbohydrate Polymers 245 (2020) 116592 T.F.S Evangelista, et al observed suggest the presence of interactions between components For instance, the two bands related to the –NH2 symmetric and asymmetric stretching were shifted to longer wavenumbers (respectively 3426 and 3356 cm−1) SSD/SPEC was also characterized by 1H NMR It is possible to observe in Fig 3b the incorporation of aromatic hydrogens at δH 8.60 to 7.00 from SSD (See Fig S5, Supporting Information), confirming the presence of the drug in the SPEC matrix Finally, a preliminary in vitro release test using dialysis membranes was performed in phosphate buffer to evaluate the role of the SPEC during the release of the drug Prior to this study, the molar extinction coefficients (ε) of SSD was calculated in phosphate buffer using the Beer-Lambert law The absorption maxima of this drug in 262 nm showed a good linear relationship to the concentration range (see Fig S6, Supporting Information) Thus, the calculated value for ε was 2.993 × 104 L mol−1 cm−1 This information is important to calculate the released amount of the drug during the in vitro release studies Fig S7 (see Supporting Information) shows the drug release profile from pure SSD and SSD/SPEC It was observed that the burst release for pure SSD occurs statistically at h, when approximately 60 % of the drug was released from the dialysis bag After that, the drug concentration is practically constant On the other hand, the release of SSD from SPEC showed a different behavior when compared to the free drug In this case, it was observed an initial slow stage (0–3 h), which may be associated with the swelling properties of the polymeric matrix (Ćirić et al., 2020) As soon as this first swelling step is overcome, the release process starts and approximately 20 % of the drug was released in h, when the release curve reaches an intermediate plateau up to 15 h After that, it is observed another increase up to 48 h, when approximately 80 % of the drug is released Comparing both systems, the drug release rate for SSD/SPEC was almost 10 times slower than pure SSD, suggesting that the incorporation of the drug on polymer matrix surface was essential to slow the drug release process Finally, the slower and continuous release rate found for SSD/SPEC can be advantageous for the design of topical drug release systems for local treatment, such as wound dressings Fig DRS spectrum of CRG, βCD-CS, and SSD/SPEC, SSD SPR band position ranged from ultraviolet to near-infrared and was strongly dependent upon their shapes and sizes The reduction of Ag+ ions can be performed via sonochemical methods in the presence of biopolymers For instance, Elsupikhe and coworkers showed a sonochemical synthesis of colloidal silver particles using κ-carrageenan (Elsupikhe, Shameli, Ahmad, Ibrahim, & Zainudin, 2015) The mechanism for the reduction of Ag+ ions was proposed by the authors and started with the generation of %H and O%H free radicals by ultrasonic irradiation prior to the reduction of Ag+ ions to Ag° TEM images of SSD/SPEC fully confirm the presence of anisotropic structures after the interaction between SSD and SPEC As observed in Fig 7a and b, the sub-micro particles shape of the SPEC is preserved after the inclusion of SSD Also, it is observed the presence of metallic silver nanostructures (in higher contrast) distributed in the polymer matrix surface However, the reduction process leads to the formation of Ag nanoparticles with irregular morphologies and large size distribution Fig 7c shows the occurrence of larger silver nanocubes, nanospheres and other morphologies off the polymeric surface (other images can be found in Fig S4, Supporting Information) Thus, as expected the size distribution for this sample is large (see Fig 7d), which explains the broad SPR band found The SSD/SPEC inclusion complex was also characterized by FTIR and 1HNMR As seen in see Fig 2, the free SSD exhibited characteristic bands at 3392, 3344, 1630, 1551, 1501, 1418, 1228 and 1125 cm−1 The peaks at 3392, 3344 and 1630 cm−1 are assigned to –NH2 symmetric and asymmetric stretching and NH2 bending, respectively The vibrational stretching of its phenyl structure conjugated to the NH2 group appears at 1551 cm−1 A band at 1500 cm−1 assigned to the phenyl skeletal vibration was also observed The peaks centered at 1228 and 1125 cm−1 are assigned to the asymmetrical stretching of the SeO bonding These results are in agreement with previous reports (Shao et al., 2017) After the incorporation of the drug into the polymeric particles, all the peaks related to SSD were found, proving the presence of this drug in the as-prepared SPEC However, some spectral changes 3.5 Theoretical study Various non-covalent interactions, such as hydrogen bonding, electrostatic forces, and π,π-stacking, can be the driving forces to the formation and stabilization of a host-guest complex (Al-Jaber & BaniYaseen, 2019; Aree & Jongrungruangchok, 2018) Due to the nature of these interactions, sometimes the analytical characterization of these supramolecular species can be a difficult task In this context, theoretical methods, such as semi-empirical PM3, are very useful tools for studying the inclusion process Various reports have shown the effective use of PM3 to calculate some energetic parameters, including thermodynamic data, as well as the geometrical structure of these inclusion complexes (de Araújo et al., 2017; Geng et al., 2018; Yang et al., 2018) The isolated host and guest and the inclusion complexes structures were fully optimized by PM3 without any symmetry constraints Herein, the chitosan or carrageenan structures were not calculated, as they not participate directly in the inclusion process Fig shows the upper and side views of all the inclusion complex optimized structures obtained by energy minimization at the PM3 level of theory To quantify the interaction between these entities in the optimized geometries, the binding energy (or complexation energy, ΔEcomplexation ) was calculated as follows: ΔEcomplexation = EβCD−SSD − (EβCD + ESSD) (3) This equation considers the difference between the heat of formation of the complex (EβCD−SSD) and the heat of formation of the free guest (ESSD ) and host (EβCD ) molecules Thus, the magnitude of the energy change is a sign of the driving force toward the inclusion complex formation As shown in Table 1, the binding energies for all the inclusion complexes were negative, indicating that the complexation Carbohydrate Polymers 245 (2020) 116592 T.F.S Evangelista, et al Fig TEM images and size distribution of SSD/SPEC antibacterial zone of inhibition for S aureus (16 ± mm) followed by K pneumoniae (13 ± mm), E coli (13 ± mm) and E durans/hirae (12 ± mm) These values are very close to the ones reported for chitosan (Bhadra, Mitra, Das, Dey, & Mukherjee, 2011; Muthuchamy et al., 2020; Nigam, Kumar, Dutta, Pei, & Ghosh, 2016), suggesting that, even after the modification, the natural antibacterial property remains active This result is expected since the substitution reaction does not consume all the amino groups, leaving the surface positively charged, as observed before The other counterpart of the SPEC, CRG, also did not present a zone of inhibition after the contact with all the bacterial strains In fact, CRG is generally used as an antibacterial agent after the incorporation of other materials (such as metal or semiconductor nanoparticles) to its structure or after chemical modifications (Zhu et al., 2017) Moderate in vitro antimicrobial activity of chitosan and βCD-CS against drug-resistant bacterial pathogens has already been reported by others (Ding et al., 2019; Verlee, Mincke, & Stevens, 2017) However, the exact antibacterial mechanism is still not fully understood, as its mode of action is significantly influenced by various factors, including the type of microorganism, the molecular weight, the degree of deacetylation, ionic strength and pH (Shahid-ul-Islam & Butola, 2019) It is usually accepted that the interaction is mostly electrostatic, occurring between the positively charged amine groups of chitosan with the negatively charged molecules from the bacterial wall or plasma membrane (Perinelli et al., 2018) Then, the permeabilization of the antimicrobial agent into the cell surface leads to leakage of intracellular constituents (such as of nucleic acids, proteins, low-molecular-weight materials), disturbing the physiological activities of the microorganisms and causing cell death processes are exothermic The complexes having the NH2 group from the drug molecule pointed toward the narrower βCD rim were found to have the smaller complexation energy (approximately −19.45 kcal/ mol for both structures), while NH2-out and NH2-c-out complexes had the highest complexation energy with βCD The energy difference between these two orientations can reach 5.63 kcal/mol It means that the NH2-in orientation is energetically preferred over the complexes which present the pyrimidine ring pointing toward the narrower rim of βCD A similar result was previously reported by our group for inclusion complexes based on sulfadiazine and hydroxypropyl-β-cyclodextrin (de Araújo et al., 2008), where the optimized NH2-in orientation was energetically favored (over the NH2-out conformation) and presented protons from the aniline ring closer to the secondary face of the cyclic oligosaccharide 3.6 Antibacterial activity assays The antimicrobial activities of the as-prepared SPEC compounds towards Gram-positive bacteria Staphylococcus aureus (ATCC 25923) and Enterococcus durans/hirae (SS1225/ IAL 03/10) and Gram-negative bacteria Klebsiella pneumoniae (ATCC 700603) and Escherichia coli (ATCC 25922) were investigated in this work These bacterial strains were chosen because they are nosocomial pathogens commonly responsible for biofilm-related infections First, the initial screening for the antimicrobial properties of the as-prepared SSD/SPEC, as well as pure βCD-CS, SSD, and GTM, were evaluated by a disc diffusion method, as presented in Table As seen, pure βCD does not show any antibacterial activity, while βCD-CS showed a clear activity on both Gram-positive and Gram-negative bacteria, presenting the highest Carbohydrate Polymers 245 (2020) 116592 T.F.S Evangelista, et al Fig Upper and side views of all SSD/SPEC inclusion complex optimized structures obtained by energy minimization at the PM3 level of theory 10 Carbohydrate Polymers 245 (2020) 116592 T.F.S Evangelista, et al concentration (MIC) values were found for SSD/SPEC and for the isolated SSD and GTM drugs (see Table 2) MIC can be defined as the minimum concentration of the antibacterial agent, which exerts the visible bacterial growth inhibition Based on the 96-well microdilution assay, it was found that both SSD/SPEC and the isolated SSD presented MIC values of 60 and 32 μg/mL for E coli and S aureus, respectively, showing that the antibacterial activity was not lost after the formation of the inclusion complex The MIC values for the antibacterial assay in the presence of GTM were around 128 and 64 μg/mL for E coli and S aureus, respectively, which is twice as higher in concentration than the test with pure SSD or SSD/SPEC Table Binding energies (kcal mol−1) obtained by PM3 for SSD/βCD inclusion complexes in different conformations Sample ΔEcomplexation (kcal mol−1) NH2-in NH2-out NH2-c-in NH2-c-out −19.4483 −13.8147 −19.4403 −17.5297 Table Zone of inhibition diameter of βCD-CS, βCD, CRG, SSD, GTM, and SSD/SPEC and Minimum Inhibitory Concentration (MIC) of SSD, SSD/SPEC and GTM against Gram-positive and Gram-negative bacterial strains Bacterial strains Conclusions In conclusion, this work showed a comprehensive description of properties, characterization and in vitro applications of supramolecular polyelectrolyte complexes based on a cyclodextrin-grafted chitosan derivative and carrageenan The formation of these SPECs was confirmed by FTIR, SEM, and ζ-potential measurements Due to the presence of inclusion sites from the attached βCD, the as-prepared materials could be suitably applied as controlled drug release systems, using silver sulfadiazine as the model drug Also, DRS and TEM analyses have shown the formation of metallic silver nanostructures via the reduction of Ag+ ions from the drug, which can improve the bacterial activity of the composite In vitro tests revealed that the as-prepared SSD-SPECs conjugated presented a clear bacterial activity toward Gram-positive bacteria Staphylococcus aureus and Enterococcus durans/hirae and Gramnegative bacteria Klebsiella pneumoniae and Escherichia coli, which was compared to gentamicin Zone of inhibition diameter (in mm) βCD-CS S aureus 16 ± E durans/hirae 12 ± K pneumoniae 13 ± E coli 13 ± MIC values (μg/mL) S aureus – E coli – βCD CRG SSD GTM SSD/SPEC 0 0 0 0 25 ± 17 ± 20 ± 23 ± 18 ± 19 ± 20 ± 18 ± 18 ± 19 ± 18 ± 17 ± – – – – 32 64 64 128 32 64 According to the CLSI interpretive criteria for disk diffusion tests (CLSI, 2019), when βCD-CS is used in our experimental condition, S aureus can be classified into the intermediate (I) category, since it presented a zone diameter between 15–19 mm Additionally, the other bacterial strains studied here were resistant (R) to this polymer, since all the zone diameters were below 14 mm The intermediate category implies clinical efficacy where the drug is concentrated in body sites Also, the I category is associated with clinical events when a higher than normal dosage of a drug can be used On the other hand, the R category implies that the drug efficiency against the microorganism has not been reliably shown in treatment studies Herein, SSD was incorporated to the as-prepared SPEC This drug is normally used as an external antibacterial agent, as in the treatment of burns, and presents a broad-spectrum activity against both Gram-positive and Gram-negative organisms As seen in Table 2, the isolated drug presented a clear bacterial activity to all the microorganisms used in this work Despite being a well-known and widely used drug, the precise antibacterial mechanism of the silver salt form of sulfadiazine is not well-clarified among the scientific community and still opened to debate It is believed that the efficacy of SSD is related to the slow and sustained delivery of silver ions, which directly attaches onto cell surfaces, damaging both cell envelope and bacterial genetic material (Munhoz, Bernardo, Malafatti, Moreira, & Mattoso, 2019) According to the CLSI interpretive criteria, S aureus, K pneumoniae and E coli were susceptible (S) to SSD, while E shown/hirae can be classified into the intermediate category The S category implies that the microorganisms were inhibited by the usually achievable concentrations of the drug, resulting in likely clinical efficacy After the incorporation of this drug into the polymeric complex, the values for the zone of inhibition diameter were very close to the isolated drug, proving the presence and the bacterial activity of SSD when associated with the SPEC Additionally, the presence of silver nanostructures (as found by DRS and TEM analyses) contributes to improving the antibacterial activity because of the slow Ag+ leaching from metallic silver, as reported by Manna and coworkers (Manna, Goswami, Shilpa, Sahu, & Rana, 2015) Also, these results were compared to gentamicin, an aminoglycoside antibiotic widely used in various types of bacterial infections As observed in Table 2, GTM exhibited similar results like the ones obtained when SSD/SPEC was used After the disc diffusion susceptibility test, the minimum inhibitory CRediT authorship contribution statement Thamasia F.S Evangelista: Methodology, Project administration, Investigation George R.S Andrade: Conceptualization, Methodology, Writing - original draft, Writing - review & editing, Visualization, Project administration, Investigation Keyte N.S Nascimento: Investigation Samuel B dos Santos: Investigation Caroline Da Ros Montes D'Oca: Investigation Charles dos S Estevam: Investigation Iara F Gimenez: Investigation Luís E Almeida: Conceptualization, Methodology, Writing - original draft, Writing - review & editing, Supervision, Funding acquisition, Investigation Declaration of Competing Interest The authors declare no competing financial interest Acknowledgments This work was financially supported by CNPq, Capes, Fapitec/SE and FAPES/ES G.R.S.A received a postdoctoral scholarship from Capes (PNPD/UFS) 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.2020.116592 References Akỗakoca Kumbasar, E P., Akduman, ầ., & ầay, A (2014) Effects of β-cyclodextrin on selected properties of electrospun thermoplastic polyurethane nanofibres Carbohydrate Polymers, 104, 42–49 https://doi.org/10.1016/j.carbpol.2013.12.065 Al-Jaber, A S., & Bani-Yaseen, A D (2019) On the encapsulation of Olsalazine by βcyclodextrin: A DFT-based computational and spectroscopic investigations Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 214, 531–536 https://doi.org/10.1016/j.saa.2019.02.030 Allen, F H (2002) The cambridge structural database: A quarter of a million crystal 11 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dropped into the chitosan solution, instantaneous... In another work, Hardy and co-workers prepared compact polyelectrolyte complexes based on βCD-functionalized chitosan/ alginate for controlled release of anti-inflammatory drugs (Hardy et al., 2018)