Chitosan – Rosmarinic acid conjugates with antioxidant, anti-inflammatory and photoprotective properties

Rosmarinic acid is an attractive candidate for skin applications because of its antioxidant, anti-inflammatory, and photoprotective functions, however, its poor bioavailability hampers its therapeutic outcome. In this context, synthesis of polymer conjugates is an alternative to enlarge its applications.

Carbohydrate Polymers 273 (2021) 118619 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Chitosan – Rosmarinic acid conjugates with antioxidant, anti-inflammatory and photoprotective properties ˜ al a, Javier Caro-Leo ´n b, Eva Espinosa-Cano a, c, María Rosa Aguilar a, c, *, Miguel Huerta-Madron a, c Blanca V´ azquez-Lasa a b c Group of Biomaterials, Institute of Polymer Science and Technology ICTP-CSIC, Madrid, Spain Grupo de Investigaci´ on en Biopolímeros, Centro de Investigaci´ on en Alimentaci´ on y Desarrollo A.C., Sonora, Mexico Networking Biomedical Research Centre in Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, Madrid, Spain A R T I C L E I N F O A B S T R A C T Keywords: Chitosan Rosmarinic acid Conjugates Antioxidant activity Photoprotective properties Wound healing Rosmarinic acid is an attractive candidate for skin applications because of its antioxidant, anti-inflammatory, and photoprotective functions, however, its poor bioavailability hampers its therapeutic outcome In this context, synthesis of polymer conjugates is an alternative to enlarge its applications This work describes the synthesis of novel water-soluble chitosan – rosmarinic acid conjugates (CSRA) that have great potential for skin applications Chitosan was functionalized with different contents of rosmarinic acid as confirmed by ATR-FTIR, 1H NMR and UV spectroscopies CSRA conjugates presented three-fold radical scavenger capacity compared to the free phenolic compound Films were prepared by solvent-casting procedure and the biological activity of the lixiv­ iates was studied in vitro Results revealed that lixiviates reduced activation of inflamed macrophages, improved antibacterial capacity against E coli with respect to native chitosan and free rosmarinic acid, and also attenuated UVB-induced cellular damage and reactive oxygen species production in fibroblasts and keratinocytes Introduction Phytochemical is a broad term meaning plant (phyto) chemical that refers to a wide variety of plant-derived compounds with beneficial therapeutic activities on human health such as anticarcinogenic, anti­ mutagenic, anti-inflammatory, and antioxidant properties (El-Sherbiny et al., 2016; Huang et al., 2016; Shahidi & Ambigaipalan, 2015; Tsao, 2010; Vuolo et al., 2019) The most common phytochemical found in human diet are polyphenols (Mrduljas et al., 2017) These compounds are one of the most widespread groups of bioactive molecules distrib­ uted almost ubiquitously in nature; they can be found in fruits, cereals, vegetables, tea, coffee and cocoa among others (Abbas et al., 2017; Shahidi & Ambigaipalan, 2015; Souto et al., 2019) It is generally accepted that the primary cause of aging and agerelated diseases as well as cancer is the cellular damage exerted by aberrant production of reactive oxygen and nitrogen species, resulting from an imbalance in cellular metabolism (Fachel et al., 2019; Vittorio et al., 2017) In this sense, the attributed anti-inflammatory, car­ dioprotective, neuroprotective, and antiaging properties of polyphenols are related to their potent antioxidant capacity which directly arises from their chemical structure (Mrduljas et al., 2017; Shahidi & Ambi­ gaipalan, 2015; Singla et al., 2019; Souto et al., 2019), can play an important role in the treatment of these pathological processes Chitosan (CS) is a cationic polysaccharide that presents many promising properties for biomedical applications such as excellent biocompatibility and biodegradability, abundance and low cost, besides other well-known biological activities: antibacterial, antifungal among ˆme, 2013; Islam et al., 2017; Rinaudo, 2006) It is others (Croisier & J´ero obtained from the alkaline deacetylation of chitin and consists of Dglucosamine and N-acetyl-D-glucosamine units linked by β-1, glyco­ sidic linkage (Muxika et al., 2017) The difference between chitin and chitosan relies on the content of acetylated groups, expressed as degree of acetylation, as well as the distribution of the acetyl groups along its structure, known as degree of acetylation These characteristics strongly affect chitosan properties and open the door to chemical modifications to broaden its application area In fact, chemical modifications of its functional groups have led to numerous useful biopolymers with different fields of application, such as cosmetics, wound healing, * Corresponding author at: ICTP-CSIC, 28006 Madrid, Spain E-mail addresses: miguel.huertam@ictp.csic.es (M Huerta-Madro˜ nal), javiercaroleon@gmail.com (J Caro-Le´ on), 100292351@alumnos.uc3m.es (E EspinosaCano), mraguilar@ictp.csic.es (M.R Aguilar), bvazquez@ictp.csic.es (B V´ azquez-Lasa) https://doi.org/10.1016/j.carbpol.2021.118619 Received 24 May 2021; Received in revised form 24 August 2021; Accepted 26 August 2021 Available online September 2021 0144-8617/© 2021 The Author(s) Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) M Huerta-Madro˜ nal et al Carbohydrate Polymers 273 (2021) 118619 pharma, biosensors, packaging or agriculture (Boeriu & van den Broek, 2019; Islam et al., 2017) Several examples of polyphenols-chitosan derivatives with physico­ chemical and biological improvements, such as superior water solubility or radical scavenger activity, can be found in the literature following different strategies (Aytekin et al., 2010; Curcio et al., 2009; Fan et al., 2017; Hu & Luo, 2016; Ilyasoglu & Guo, 2019; S Kim, 2018; Vittorio et al., 2017; Xu et al., 2015) Polyphenols have been grafted into chi­ tosan backbone through several techniques including enzyme-mediated modification, free radical induced grafting reaction and activated estermediated modification (Hu & Luo, 2016) Polyphenol oxidases, as tyrosinase and laccase, are able to convert phenols in highly reactive species that covalently bind to chitosan amine groups The free radical induced grafting method involves the use of a redox pair to generate hydroxyl chitosan radicals in which polyphenols are inserted In acti­ vated ester-mediated modification, different coupling agents have been used to covalently conjugate a phenolic acid to chitosan Among them, the most widely used coupling agent to link phenolic carboxylic groups to amine moieties of chitosan is the water-soluble 1-ethyl-3-(3-dimethy­ laminopropyl)carbodiimide (EDC) Rosmarinic acid (RA) corresponds to the hydroxycinnamic acid family and it is an ester of 3,4-dihydroxyphenyllactic acid and caffeic acid (Silveira Fachel et al., 2019; Fadel et al., 2011) It is a ubiquitous phenolic compound found in more than 30 families of plants with many remarkable biological and pharmacological activities The well-known antioxidant potential of RA (Amoah et al., 2016; Fadel et al., 2011; Kim et al., 2015; Qiao et al., 2005; Tache et al., 2012), consequence of the two catechol groups present in its structure, give rise to other extensively studied biological properties such as anti-inflammatory (Amoah et al., 2016; Luo et al., 2020; Qiao et al., 2005), antiviral (Amoah et al., 2016; Kim et al., 2015), antitumoral (Amoah et al., 2016; Fachel et al., 2019), neuroprotective (Amoah et al., 2016; Fachel et al., 2019; Silveira Fachel et al., 2019), photoprotective (Cutrim & Cortez, ´nchez et al., 2016), and wound 2018; Osakabe et al., 2004; P´ erez-Sa healing (Amoah et al., 2016; Chhabra et al., 2020; Küba et al., 2020; Wani et al., 2019) These characteristics have led to its pharmaceutical and analytical development as a natural molecule of interest in biomedical applications For example, in skin applications, topical or local delivery of rosmarinic acid has shown potential to reduce the risk of skin cancer preventing tissue damage by oxidative stress, and to accelerate wound healing in murine models (Chhabra et al., 2020; Hossan et al., 2014; Küba et al., 2020; Osakabe et al., 2004; Wani et al., 2019) However, its poor bioavailability due to high instability, ineffi­ cient permeability through biological barriers and poor water solubility hamper its therapeutic outcome (Amoah et al., 2016; Fachel et al., 2019; Kim et al., 2015) In this context, nanotechnology-based drug delivery systems have been proposed to overcome these limitations (Chhabra et al., 2020; da Silva et al., 2016; Kuo & Rajesh, 2017; Vittorio et al., 2017; Wani et al., 2019) RA encapsulation in nanostructures has been proved to allow a spatio-temporal controlled release increasing its bioavailability while reducing the cytotoxicity effects (Baptista da Silva et al., 2014; Bastos et al., 2016; da Silva et al., 2016; Fachel et al., 2019) Another strategy to solve low bioavailability issues, consists on the synthesis of polymer conjugates composed of a drug covalently linked to a macromolecular system to develop a high effective therapy using the favourable biological properties of polyphenols (Aytekin et al., 2010; Hu & Luo, 2016; Kim, 2018; Pokhrel & Yadav, 2019; Ryu et al., 2011; Xu et al., 2015) This approach can be also exploited to prepare RA conju­ gates with different polymers (Calzoni et al., 2019; Ge et al., 2018; Parisi et al., 2017) that may overcome bioavailability issues and adverse ef­ fects of free administered rosmarinic acid In this context new materials with multiple bioactivities to stimulate wound healing or protect skin from exogenous damage are being sought Our hypothesis establishes that the chitosan-RA conjugate (CSRA) could give rise to a new material with properties already described for rosmarinic acid (i.e antioxidant, anti-inflammatory, and photoprotective) and for chitosan (i.e antimicrobial activity and biodegradability) that would benefit in skin applications Materials and methods 2.1 Synthesis and characterization of rosmarinic acid-chitosan conjugates Rosmarinic acid (RA, 96% pure, Merck KGaA, Darmstadt, Germany) was conjugated to chitosan (CS, 90/200, 90% degree of deacetylation, viscosity 151–350 mPas (1% in 1% acetic acid, 20◦ )) (Chitoscience, Halle, Germany) backbone by carbodiimide coupling using (1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), commer­ cial grade, Merck KGaA, Darmstadt, Germany) as a coupling agent Reaction was performed at pH 5.0 and room temperature, as reported by Aytekin and colleagues (Aytekin et al., 2010), avoiding light exposure to minimize phenol oxidation Briefly, chitosan (290 mg) was dissolved in a mixture of 2.5 mL acetic acid (0.1%) (Merck KGaA, Darmstadt, Ger­ many) solution and 22.75 mL Milli-Q water The resulting solution was adjusted to pH 5.0 with addition of NaOH (0.2 M) (Merck KGaA, Darmstadt, Germany) dropwise Different CS:EDC:RA molar ratios were used in order to obtain conjugates with varying RA content Reacting mixture was left under stirring overnight at room temperature Sepa­ rately, RA and EDC were dissolved in 13.5 mL ethanol (Merck KGaA, Darmstadt, Germany) and 17 mL Milli-Q water respectively and then added drop-by-drop to the chitosan solution Afterwards, pH was read­ justed to 5.0 with NaOH (0.2 M) and mixture was stirred for h at room temperature and in darkness At the end of the reaction, the resultant solution was dialyzed (dialysis membrane MWCO 3.5 kDa, Merck KGaA, Darmstadt, Germany) against acid Milli-Q water (pH adjusted to 5.0) for days and Milli-Q water for another 24 h to eliminate rests of RA, NaOH and isourea Upon dialysis, the solution was frozen and lyophilized to obtain CSRA conjugates as yellow powders which were stored at ◦ C and avoiding light exposure until used In the present paper CSRA conjugates will be designated as CS-XRA, X being the effective per­ centage of rosmarinic acid in chitosan polysaccharide rings obtained by UV spectroscopy (see code samples in Table in Subsection 3.1.3) 2.2 Physicochemical characterization 2.2.1 ATR-FTIR spectroscopy ATR-FTIR spectra of lyophilised CSRA conjugates were recorded in the mid-infrared absorbance region (4000–1000 cm− 1) using a PerkinElmer (Spectrum One) spectrometer equipped with an ATR accessory using 32 scans and a resolution of cm− 2.2.2 1H NMR spectroscopy H NMR spectra were recorded in a Varian Mercury equipment operating at 500 MHz at 45 ◦ C in presaturated conditions Conjugates and native chitosan were dissolved in a 49:1 (v/v) solvent mixture deuterium oxide (D2O, Merck KGaA, Darmstadt, Germany):deuterium chloride (DCl, Merck KGaA, Darmstadt, Germany) at 25 ◦ C RA was dissolved in deuterated DMSO (DMSO‑d6, Merck KGaA, Darmstadt, Germany) Spectral analysis and proton identification were performed using MestreNova 9.0 2.2.3 UV spectrophotometry UV spectra of conjugates and RA dissolved in acetic acid (0.1%) were recorded at 25 ◦ C using a NanoDrop One spectrophotometer (Thermo Fisher Scientific) to determine the degree of conjugation of RA in the corresponding conjugate 2.2.4 TGA TGA diagrams were obtained in a thermogravimetric TGA Q500 (TA instruments) apparatus Samples were analysed in a range of 30–600 ◦ C under nitrogen at a heating rate of 10 ◦ C/min Maximum thermal M Huerta-Madro˜ nal et al Carbohydrate Polymers 273 (2021) 118619 Table Sample codes, CS:EDC:RA feed molar ratio x 103, theoretical and effective percentage of rosmarinic acid conjugated to each CSRA (*obtained by UV spectrophotometry (λ = 325 nm), and effective RA:CSRA mass ratio (μg:mg) of conjugates Sample CS-10RA CS-5RA CS-0.8RA CS-0.4RA CS:EDC:RA feed molar ratio × 103 1.580:0.449:0.766 1.580:0.449:0.383 1.580:0.449:0.192 1.580:0.449:0.096 RA conjugation (%) Effective RA:CSRA mass ratio (μm:mg) Theoretical Effective* 48 24 12 10.0 5.0 0.8 0.4 decomposition temperature (Tmax) as well as weight loss and residue, both at 600 ◦ C, were calculated from TGA and derivative curves (DTG), respectively Each experiment was repeated three times for each sample 173.5 99.0 17.5 8.0 polyphenol was dissolved in DMEM without phenol red (250 μg/mL) Serial dilutions were performed to obtain the different RA concentra­ tions and pH was measured (pH 7) Likewise, RSA of films lixiviates with different RA concentrations (Table S1) were tested RSA was obtained following the protocol described above for RA and CSRA conjugates All results are given as mean ± SD (n = 8) 2.3 Film preparation and release kinetics Thin films (around 200 μm thickness) of the CSRA samples were obtained by a solvent casting methodology; 2.5 mL of a Milli-Q water solution of the corresponding conjugate polymer (2.5 mg/mL) was poured to a P12 glass plate (22.4 mm diameter) at room temperature Films were left to dry at room temperature avoiding exposure to light Release kinetics were obtained by immersion of the corresponding conjugate film in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) without phenol red (Gibco, Waltham, MA USA) at 37 ◦ C Lix­ iviates were collected at several time points for days Appropriate calibration curves of RA in DMEM without phenol red (2.5–30 μg/mL) with R2 = 0.9997 (Abs (325 nm) = 0.0397*[RA] (μg/mL) + 0.0188) were prepared in order to determine the amount of catechol species released from each sample Each experiment was repeated three times and results are given as mean ± standard deviation (SD) Lixiviates collected in DMEM were used to analyse the effect of the conjugates in the free radical and reactive oxygen scavenger capacity (see Subsections 2.4 and 2.5.3, respectively), and in the cytotoxicity, nitric oxide reduction, and photoprotective assays (Subsection 2.5) For the antibacterial capacity assay, the corresponding conjugate film was immersed in bacteria culture broth (Merck KGaA, Darmstadt, Germany) and lixiviates collected at h were used in these experiments (Subsec­ tion 2.5.4) 2.5 Cell culture experiments Murine macrophages (RAW 264.7) and human dermal fibroblasts (FBH) cell lines were purchased from Merck (Merck KGaA, Darmstadt, Germany) Human epidermal keratinocytes (HEK) cell line was obtained from Innoprot (Derio, Bizkaia, Spain) RAW 264.7 and FBH cells were maintained over permissive conditions in high-glucose DMEM supple­ mented with 10% Fetal Bovine Serum (FBS) (Gibco Waltham, MA USA), 2% L-Glutamine (Merck KGaA, Darmstadt, Germany) and Penicillin-G (Merck KGaA, Darmstadt, Germany) at 37 ◦ C in a humidified incu­ bator with 5% CO2 The HEK cell line was cultured in the Keratinocytes Medium Kit from Innoprot (Derio, Bizkaia, Spain) and maintained over permissive conditions in a humidified incubator with 5% CO2 In vitro cell culture experiments were performed with lixiviates of CSRA film samples collected in DMEM at h at 37 ◦ C (Table S1) and after sterili­ zation by 0.22 μm polyether sulfone (PES) filtration (Merck KGaA, Darmstadt, Germany) before use 2.5.1 Cytotoxicity assay In order to evaluate the toxicity of the CSRA film lixiviates Alamar Blue Reagent (AB, Invitrogen) was used to determine cell viability RAW 264.7, FBH and HEK were seeded in 96 well-plates under permissive conditions at 200,000, 90,000 and 100,000 live cells/mL (100 μL per well) After 24 h, cells were treated with either fresh DMEM (as positive control) or the corresponding lixiviate sample Then, upon 24 h of exposure to lixiviates, cellular viability was determined using AB Absorbance at 570 nm was measured by a Multi-Detection Microplate Reader Synergy HT (BioTek Instruments) Percentage of cell viability was expressed with respect to the positive control (fresh DMEM) All results are given as mean ± SD (n = 16) 2.4 Radical scavenger activity Radical scavenger activity (RSA) of CSRA conjugates was determined by measuring the decolorization of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) (Merck KGaA, Darmstadt, Germany) from the trapping of its unpaired electron, according to the method reported by Qiao et al (2009) with slight modification In addition, RSA of free RA was eval­ uated for comparison purposes Stock solutions of RA (25 μg/mL) and CSRA conjugates (1000 μg/mL) were prepared in acetic acid (0.1%) (pH 4) and successively diluted Serial dilutions containing different con­ centrations of free RA and polymer conjugate were tested Briefly, 100 μL DPPH ethanol solution (0.25 mM) were added to 100 μL of the cor­ responding CSRA conjugate sample, lixiviate or free RA Mixture was allowed to react under stirring for 30 in dark and room temperature conditions Then, the absorbance was measured at 515 nm against a blank (100 μL of acetic acid solution (0.1%) and 100 μL of DPPH solu­ tion) with a Multi-Detection Microplate Reader Synergy HT (BioTek Instruments; Vermont, USA) The RSA was calculated as: radical scav­ enging capacity (%) = (((A0 − (A1 − A2)) / A0) * 100, where A0 is the absorbance of the blank, A1 is the absorbance of the sample and A2 is the absorbance of the sample under identical conditions as A1 with ethanol instead of DPPH solution Therefore, the smaller the absorbance of the mixture, the higher the radical scavenger activity of the tested sample RSA was expressed in percentage and results are given as mean ± SD (n = 16) To study the influence of pH in the RSA of rosmarinic acid, the 2.5.2 Nitric oxide release assay The anti-inflammatory activity of CSRA conjugate lixiviates was investigated using nitric oxide (NO) assay RAW 264.7 cells were seeded in a 96 well-plate under permissive conditions at a concentration of 200,000 live cells/mL (100 μL per well) After seeding and 24 h incu­ bation, cells were treated with only DMEM (negative control), DMEM with μg/mL lipopolysaccharide (positive control) (LPS, from E coli O111:B4; CAS Number: 297-473-0, Merck KGaA, Darmstadt, Germany) or non-toxic CSRA lixiviates with μg/mL LPS Upon 24 h of treatment, LPS-induced NO release was measured for each condition using Griess Reagent kit (Merck KGaA, Darmstadt, Germany) following manufac­ turer specifications Absorbance was measured at 540 nm by a MultiDetection Microplate Reader Synergy HT, and data were expressed as percentage of NO production with respect to the positive control (100% NO production) Results are given as mean ± SD (n = 16) M Huerta-Madro˜ nal et al Carbohydrate Polymers 273 (2021) 118619 2.5.3 UVB irradiation and reactive oxygen species assay For the UV irradiation, an Ultraviolet Crosslinker (Model CL-1000 L, UVP) with a bank of × 0.4 mW/cm2 tubes was used The emission spectrum was in the UVB range (280–320 nm) with an emission peak at 313 nm RAW 264.7, HFB and HEK were seeded in 96-well plates at 150,000, 90,000 and 100,000 live cells/mL respectively, and main­ tained in medium for 24 h For the treatment, cells were PBS-washed and covered with a thin layer (50 μL) of DMEM without phenol red (positive control) or CSRA lixiviates Well plates were placed at 25 cm from the lamps and the irradiation dose consisted of one single pulse of 100, 140 and 140 mJ/cm2 for RAW 264.7, HFB and HEK, respectively Nonirradiated cells (negative controls) were treated similarly and covered with a black panel barrier to eliminate unnecessary stimulation After­ wards, the medium was replaced with fresh media and cells were either incubated for 24 h for the viability assay (see Subsection 2.5.1) or treated with the H2DCF-DA probe, after h incubation in case of HFB and HEK, or 24 h incubation in case of RAW 264.7 for intracellular reactive oxygen species (ROS) imaging Percentage of cell viability after UV irradiation was expressed with respect to non-irradiated cells and results are given as mean ± SD (n = 24) The total ROS free radical activity was fluorometrically measured using 2′ ,7′ -dichlorofluorescin diacetate (H2DCF-DA, Merck KGaA, Darmstadt, Germany) After UVB irradiation and incubation, the cell medium was removed and 100 μL/ well of a 20 μM H2DCF-DA solution in PBS was added to the cells Then, cells were incubated at 37 ◦ C in dark conditions for 20 and washed twice with PBS before imaging Rosmarinic acid is a phenolic compound with remarkable biological activities and well-known antioxidant potential that have been used lately in its free form, encapsulated in different drug delivery systems or conjugated to several polymers (i.e gelatin, poly(lactic-co-glycolic acid) or dextran) to fully exploit its pharmacological potential (Calzoni et al., 2019; Ge et al., 2018; Parisi et al., 2017) However, to the best of our knowledge, derivatives of chitosan and rosmarinic acid have not been published yet, so, our goal was to prepare and characterize for the first time, chitosan-RA conjugates that combine the properties of each indi­ vidual component in a novel functionalized polymer 3.1 Chitosan – Rosmarinic acid conjugate synthesis and characterization CSRA conjugates with varying composition were successfully syn­ thetized via carbodiimide coupling in a one-step reaction EDC activates RA carboxylic groups to form an O-acylisourea intermediate which will couple to primary amines of chitosan via amide bond formation No precipitates or drastic pH changes were observed during the whole process After dialysis and lyophilisation, the product had a light-yellow colour consequence of the presence of RA elucidating a positive chitosan derivatization The more RA reacted with chitosan, the higher colour intensity showed the resulting polymer ATR-FTIR and 1H NMR spectra further confirmed RA inclusion into chitosan backbone 3.1.1 ATR-FTIR analysis Spectra of native and functionalized chitosan are compared in Fig S1 The characteristic chitosan pattern was observed in all spectra: N–H and O–H stretching vibrations at 3370 cm− 1, C–H stretching vibration of methylene at 2870 cm− 1, N–H bending vibration at 1600 cm− in the CS spectrum, shifted to 1605 cm− in the CSRA sample spectra, and C–O stretching involved in chitosan skeleton vibration at 1070 cm− (Tan et al., 2018) (Sajomsang et al., 2009) In addition, chitosan derivatives spectra showed the typical vibrational bands of rosmarinic acid between 1700 and 1000 cm− (Stehfest et al., 2004): a band at 1690 cm− attributed to CO stretching vibration in associated ester groups which increased with content of conjugated RA, two bands at 1605 and 1520 cm− attributed to aromatic ring stretching and two other signals at 1380 cm− and 1160 cm− due to O–H and C–O stretching, respectively All these bands overlapped with those of chi­ tosan, except for the 1520 cm− peak which confirmed RA conjugation in CSRA samples In addition, another band at 1260 cm− attributed to C–N stretching vibrations (amide III) was observed in the conjugate sample spectra that may result from the new amide bond formation (Singh, 1999) Therefore, it can be said that successful chitosan func­ tionalization is validated and supported by the characteristic chitosan peaks together with bands observed at 1520 and 1260 cm− 1, attributed to RA and amide III of the newly formed amide bond, respectively 2.5.4 Antibacterial capacity E coli (CECT DH5α) and S epidermidis (CECT 232T) were obtained from the Spanish Type Culture Collection (CECT) LB Broth and bacte­ riological agar were purchased from Merck (Merck KGaA, Darmstadt, Germany) Bacterial density was standardized to OD (optical density) value by using NanoDrop One spectrophotometer (ThermoFisher Sci­ entific) at 600 nm wavelength Dynamic growth of bacteria in the presence of CSRA lixiviates was evaluated by obtaining the OD at 600 nm after 24 h incubation, following a previously described method (Matejczyk et al., 2018) Briefly, Gram-negative E coli and Grampositive S epidermidis bacteria were seeded initially at 0.1 OD and their respective bacterial cell density measured after 24 h of incubation in the presence of either free RA, or native CS, CS-0.8RA and CS-0.4RA lixiviate samples collected in broth culture, or bacterial growth media All samples were compared to bacteria incubated under permissive conditions in bacterial growth media (negative control) The determi­ nation of bacterial growth inhibition (GI) was obtained as GI (%) = ODcontrol (%) − ODsample (%), where ODcontrol was the bacterial density of the control sample (negative control), which was equal to 100%, and ODsample corresponded to the decrease in optical density of bacteria in the presence of studied samples with respect to the ODcontrol value Gentamicin (Acofarma, Madrid, Spain) and Ampicillin (Merck KGaA, Darmstadt, Germany) were used as growth inhibition controls (positive controls) for E coli and S epidermidis respectively, and results are given as mean ± SD (n = 16) 3.1.2 1H NMR analysis To further confirm rosmarinic acid conjugation into chitosan back­ bone, 1H NMR analysis of derivatives and initial RA was conducted and main signals of chitosan and the polyphenolic compound were identified and assigned Spectrum of RA showed the typical resonance signals described in literature (Charisiadis et al., 2012) (Fig S2) All CSRA sample spectra exhibited the characteristic chitosan pattern (Sajomsang et al., 2009) (Lavertu et al., 2003) (Fig 1): a singlet at 5.2 ppm due to anomeric proton, a multiplet between 4.2 and 3.2 ppm corresponding to protons H3-H6 of the polysaccharide ring, and two singlet signals at 3.2 and 2.1 due to H2 proton of the amine group (H-amine) and the acetyl group protons (H-Ac), respectively Furthermore, 1H NMR spectra of CSRA derivatives showed a broad multiplet signal between 7.5 and 6.7 ppm assigned to aromatic protons (e–j) of conjugated RA that was shifted to lower field respect to that in the RA spectrum (between 6.4 and 5.5 ppm); a signal due to proton m of RA (7.7 ppm), a signal attributed to proton n of RA (6.5 ppm), and a triplet signal (proton k) slightly displaced and overlapped with the chitosan H2 signal at 3.2 ppm 2.5.5 Statistical analysis Statistical analysis (ANOVA) with a significance level of *p < 0.05 between controls and samples or #p < 0.05 among samples was per­ formed using Origin Pro software (Origin Lab, USA) and Tukey grouping method Results and discussion Due to an increasing need of functionalized polymers, in the past years several conjugates of chitosan and different polyphenols have been reported with improved properties such as superior water solubility, increased biocompatibility or higher radical scavenger activity (Hu & Luo, 2016; Ilyasoglu & Guo, 2019; Kim, 2018; Xu et al., 2015) M Huerta-Madro˜ nal et al Carbohydrate Polymers 273 (2021) 118619 Fig 1H NMR spectra of native chitosan (CS) and CSRA samples in a 1:49 (v/v) D2O:DCl solvent mixture at 45 ◦ C CSRA samples were named as CS-XRA, where X denotes the effective percentage of RA conjugation into chitosan determined by UV spectroscopy analysis consequence of amide bond formation crystallinity and looseness of packing structure This translates into the lower thermal stability as a result of the grafting process produced in the CSRA conjugates 3.1.3 UV spectroscopy analysis Rosmarinic acid UV spectrum shows maximum absorbance at 325–330 nm wavelength as already described (Saltas et al., 2013) Therefore, absorbance at that specific wavelength was used to determine RA concentrations in chitosan derivatives It is worth mentioning that chitosan is a polysaccharide obtained from the partial deacetylation of chitin Due to chitosan structure, RA can only be conjugated to the primary amine group of the N-glucosamine rings The percentage of chitosan polysaccharide rings to which RA was conjugated (i.e % RA conjugation) was calculated for all chitosan derivatives which were named as CS-XRA, where X denotes the effective percentage of RA conjugated to chitosan Codes of samples and results of CSRA compo­ sition are shown in Table 3.2 Release kinetics of CSRA conjugate films Release of catechol-bearing species from film samples was analysed in culture media (DMEM) without phenol red at 37 ◦ C Given that chi­ tosan derivatization was achieved via amide bond formation, as confirmed previously by ATR-FTIR and 1H NMR spectroscopies, and amide bonds are highly stable linkages resistant to hydrolysis at physi­ ological pH and body temperatures, it is very unlikely that RA molecules will be released from films to the culture media In fact, reflux, high temperatures and strong acid or basic solutions are used for its cleavage (Mahesh et al., 2018; Ouellette & Rawn, 2018; Pill et al., 2019) Because of this, lixiviates from films will be composed of catechol species most likely consisting of chitosan molecules with RA attached via their pri­ mary amine groups Fig shows the release profiles of catechol-bearing species of each sample at different time points All curves followed a similar pattern: a first initial burst release and a controlled release pattern approaching to a plateau of RA-bearing species release They showed that CS-0.4RA and CS-0.8RA reached a plateau within the first hour reaching final concentrations of 11 and 28 μg/mL (49.4 ± 2.8% and 65.9 ± 2.4% of the initial RA content) respectively, while in the case of CS-5RA and CS-10RA conjugates, both showed maximum release at h achieving 115 μg/mL and 316 μg/mL (41.7 ± 1.6% and 57.7 ± 1.5% of the initial RA amount) correspondingly 3.1.4 Thermal stability study Fig S3 shows the thermogravimetric (A) and derivative thermog­ ravimetry (DTG) (B) curves of CSRA conjugates compared to those of initial chitosan and RA Weight loss of chitosan underwent in two steps The first step observed below 150 ◦ C might be consequence of water molecules entrapped into the carbohydrate chains (Tan et al., 2018) and corresponded to 6.1% of weight loss The second step and main degra­ dation stage, in which chitosan decomposition and scission of the polymer chain occurred (Jana et al., 2015), went from 260 ◦ C (onset) to 400 ◦ C with a 69.5% weight loss at 600 ◦ C (Table S2) DTG analysis showed a maximum thermal decomposition temperature (Tmax) at 309 ◦ C for native chitosan Similarly, CSRA derivatives presented a first step of weight loss (in the range 5.0–6.8%) consequence of water evaporation, however, CSRA derivatives started to degrade at lower temperature than that of chitosan The main thermal degradation event started at 200 ◦ C and prolonged up to 400 ◦ C with a weight loss at 600 ◦ C in the range of 62.3–65.5% DTG thermographs showed that the highest decomposition rate (Tmax) arose in the range of 245–247 ◦ C for all CSRA derivatives Grafting of RA, as occur when other polyphenols are con­ jugated to chitosan (Hu & Luo, 2016), may cause a disruption of chi­ tosan intermolecular hydrogen bonds, resulting in a remarkably reduced 3.3 Antioxidant capacity 3.3.1 CSRA conjugates The radical scavenging activity of antioxidants against species such as 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), 2,2′ -azino-bis(3-eth­ ylbenzothiazoline-6-sulfonic acid) (ABTS) or the superoxide anion radical (O2⋅-) is currently measured to study the capacity of molecules to act as free radical terminators or hydrogen donors (de Vega et al., 2020; Ji et al., 2019) Of these methods, DPPH radical scavenger activity is M Huerta-Madro˜ nal et al Carbohydrate Polymers 273 (2021) 118619 Table EC50 values of CSRA conjugates and calculated RA concentrations corresponding to them Sample CSRA EC50 (μg/mL) [RA] for CSRA EC50 values (μg/mL) CS-10RA CS-5RA CS-0.8RA CS-0.4RA 16 38 210 370 2.8 3.7 3.6 2.9 pKa4 = 10.62 (Danaf et al., 2016), which probably will burden its antioxidant capacity at physiological pH In order to investigate this effect, initially the RA radical scavenger activity was evaluated at pH and and results are represented in Fig It can be observed that RSA notably reduced at pH compared to pH in the concentration range between and 250 μg/mL Separately, DPPH scavenger activity of film lixiviates obtained at pH was evaluated and results were compared respect to DMEM without phenol red alone (control) in Fig 4B Inter­ estingly, any CSRA conjugate lixiviate exerted similar antioxidant properties giving RSA values around 40% independently of the degree of functionalization Therefore, it can be said that at pH antioxidant properties of RA may be hampered caused by deprotonation of one of its catechol motives due to the proximity to pKa2 = 8.36 (Danaf et al., 2016) Fig RA-bearing species release profiles of CSRA films in culture medium at 37 ◦ C widely used as a rapid, simple and inexpensive method In this work, antioxidant capacity of CSRA samples was initially studied versus con­ centration and compared to that of free RA, whose potent radical scavenger has been reported by different authors (Adomako-Bonsu et al., 2017; Ji et al., 2019; Zhu et al., 2014) (Fig 3) In this work, for rosmarinic acid a half-maximum effective concentration (EC50) of 9.6 μg/mL (26.6 μM) was determined (Fig 3A) As it can be noticed in Fig 3B, native chitosan exerted no antioxidant capacity at any con­ centration while for the different conjugates, the higher the RA content, the higher the antioxidant capacity EC50 values of conjugates were obtained from the curves represented in Fig 5B and they are summa­ rized in Table along with the free RA concentrations corresponding to these CSRA EC50 values Interestingly, it can be observed that RA immobilization into the chitosan backbone improves its antioxidant activity, as free rosmarinic acid presents an EC50 value of 10 μg/mL while CSRA EC50 concentrations correspond to 2.8–3.7 μg/mL of free RA Therefore, CSRA presents an antioxidant activity significantly higher than free RA 3.4 Cytotoxicity of CSRA conjugates Cytotoxicity of CSRA lixiviates was monitored in HFB, HEK and RAW 264.7 cultures under ISO 10993-5:2009 and results are shown in Fig 5A–C respectively As can be observed in the graphs, lixiviates from CS-5RA and CS-10RA films resulted toxic for all three cell lines How­ ever, lixiviates from the CS-0.4RA and CS-0.8RA samples showed absence of cytotoxicity Biocompatibility of free RA was also assessed in the three cell lines in the concentration range of those of lixiviates (Fig 5D–F) The selected concentrations for free RA were the results of a wider experiment in which a larger range of concentrations were tested Those presented in the manuscript are the ones that allow determining the EC50 easier Free RA displayed cytotoxic effects at the highest concentrations, showing viability values lower than 70% at 50 μg/mL for HFB and at 75 μg/mL for both HEK and RAW 264.7 lines Interest­ ingly, similar cell viability (around 100%) were observed in the three cell lines when compared lixiviates of the CS-0.8RA and CS-0.4RA samples with their equivalent free RA concentrations (i.e., 28 and 11 μg/mL free RA respectively) 3.3.2 Influence of pH on antioxidant capacity RA excellent antioxidant properties are mainly attributed to their two catechol groups However, they present several dissociated forms depending on pH According to Danaf et al., RA catechol groups pro­ gressively lose their hydrogen atoms as pH increases from pKa1 = 2.92 to Fig Radical scavenger activity of (A) free RA and (B) CSRA derivatives and native chitosan versus concentration M Huerta-Madro˜ nal et al Carbohydrate Polymers 273 (2021) 118619 Fig (A) DPPH scavenger capacity of rosmarinic acid samples at different concentrations and at pH or and (B) CSRA lixiviates at pH = Results are the mean ± SD (n = 8) Panel B includes the ANOVA results (*p < 0.05) comparing samples against DMEM without phenol red (control) Fig Cell viability human epidermal fibroblasts (HFB), human epidermal keratinocytes (HEK) and murine macrophages (RAW 264.7) exposed to CSRA lixiviates (A–C) or and free RA (D–F) The diagrams include the mean, SD (n = 16), and the ANOVA results (*p < 0.05 statistically significant difference between the cells in DMEM (control) and treated cells, and #p < 0.05 between the cells treated with different CSRA conjugates or RA concentrations (brackets)) 3.5 Anti-inflammatory capacity were tested in a previous experiment, however, they were not included in the anti-inflammatory test since they showed to reduce RAW viability below 50% Almost null NO release was observed in non-stimulated cells (negative control) Only μg/mL of RA was enough to reduce nitric oxide production in half compared to the positive control (Control + LPS) (Fig 6A) Likewise, lixiviates of both conjugates reduced NO levels below 40%, showing similar effects than their corresponding free RA concentrations, which confirms our initial hypothesis CSRA derivatives with higher grafting of RA suppressed in a greater manner NO release, however no significant differences were observed among the two of them (Fig 6B) The anti-inflammatory capacity of biocompatible lixiviates (i.e CS0.8RA and CS-0.4RA samples) was assessed by means of the NO release assay in macrophages RAW 264.7 cell line Also, RA ability to reduce LPS-induced nitric oxide levels was assessed and used for comparative purposes Different authors already proved the capacity of rosmarinic acid and catechol bearing formulations to attenuate nitric oxide production after activation with LPS (Silveira Fachel et al., 2019; Puertas-Bartolom´ e et al., 2018; Qiao et al., 2005) Fig shows the total amount of NO released by LPS-stimulated cells expressed in percentage after treatment with RA samples at different concentrations (Fig 6A) or lixiviate samples (Fig 6B) RA concentrations higher than 50 μg/mL M Huerta-Madro˜ nal et al Carbohydrate Polymers 273 (2021) 118619 Fig Nitric oxide release by RAW 264.7 macrophages after 24 h with: (A) treatment with LPS (positive control) and different rosmarinic acid (RA) concentrations, and (B) treatment with LPS (positive control), no treatment (negative control), and treatment with LPS and h lixiviates of CS-0.4RA and CS-0.8RA The diagrams include the mean, SD (n = 16), and the ANOVA results (*p < 0.05 statistically significant difference between positive control and tested samples, and #p < 0.05 between untreated cells and cells treated with different CSRA conjugates or RA concentrations (brackets)) 3.6 Photoprotective capacity of CSRA conjugates contributions at this wavelength, and therefore if considered, they would provide incorrect RA growth inhibition values Fig shows the growth inhibition capacity of the different samples compared to nega­ tive control (incubated bacteria under permissive conditions in bacterial growth media) The obtained data revealed that RA had stronger bactericidal effect against S epidermidis than to E coli CSRA conjugates presented similar bacterial inhibition against S epidermidis than their corresponding free RA concentrations (1 μg/mL and 0.3 μg/mL, for CS0.8RA and CS-0.4RA, respectively) Nevertheless, in the case of E coli, both CSRA showed a two-fold GI value when compared to free RA Therefore, we conclude that CSRA derivatives elicited interesting bactericidal effect similar to that of native chitosan and corresponding concentration of free RA in the case of S epidermidis Noteworthy, a slight synergistic effect was shown in the case of CS-0.8RA against E coli, since higher GI can be seen when it is compared to unmodified CS, and free RA's growth inhibition capacity is doubled In the same way against E coli, superior bactericidal effect of CS-0.4RA was evidenced, however, no significant differences were observed when it was compared to native CS Rosmarinic acid has shown photoprotective capacity, increasing the cellular viability and reducing the oxidative stress of UV-irradiated cells (Fernando et al., 2016; Lembo et al., 2014; Osakabe et al., 2004; P´ erezS´ anchez et al., 2014; P´erez-S´ anchez et al., 2016) In order to study the photoprotective ability of CSRA polymers, lixiviates from biocompatible conjugates were tested to evaluate their capacity to increase cell viability and attenuate UVB-induced ROS effects after irradiation After UVB exposure, cells treated with CS-0.8RA or CS-0.4RA showed a sig­ nificant increase in the percentage of cell viability with respect to pos­ itive control (untreated and irradiated cells) (Fig 7A–C) Interestingly, a greater photoprotection was achieved with the polymer conjugate incorporating the highest amount of RA Moreover, Fig 7D–G shows results on the ROS production of HFB, HEK and RAW 264.7 upon UVB irradiation in the presence or absence of CSRA Irradiated cells without CSRA conjugates were taken as 100% of ROS production As it can be observed, due to cellular basal metabolism, a certain fluorescence signal is emitted in non-irradiated cells (negative control) When cells were exposed to UVB in the presence of CSRA lixiviates, the production of intracellular ROS was similar to the negative control Notably again, the chitosan derivative with the highest RA content was capable of reducing in a greater manner UVB-induced radical oxygen species production This fact also supports our initial hypothesis, since it proves that chi­ tosan conjugates maintained the photoprotective capacity of RA Conclusions Chitosan functionalization with RA was successfully carried out as confirmed by an extensive physicochemical characterization including ATR-FTIR, 1H NMR, UV spectroscopy and TGA analysis The resultant water-soluble conjugates have demonstrated a three-fold increase in radical scavenger activity and improved antimicrobial properties over free RA Moreover, they attenuated inflammatory activation of macro­ phages and reduced UVB-induced damage and ROS production in fi­ broblasts, keratinocytes and macrophages Altogether these data confirm our working hypothesis proving that the novel CSRA conjugates present the bioactivity attributed to the original compounds (i.e chito­ san and rosmarinic acid), as demonstrated in vitro using skin-derived cell cultures CSRA conjugates possess desirable properties for skin appli­ cations such as the treatment of age-related diseases and healing of chronic wounds 3.7 Antibacterial activity The antibacterial properties of chitosan are extensively reported in the literature since its broad-spectrum of antibacterial activity was first explained by Allan and Hardwiger (Jana & Jana, 2019) Since then, several mechanisms of antibacterial action for chitosan have been pro­ posed, however, the topic is still a matter of discussion On the other hand, rosmarinic acid antibacterial activity has already been reported by several authors (Abedini et al., 2013; Adamczak et al., 2019; Matejczyk et al., 2018; Nieto et al., 2018) as well as the synergistic bactericidal effect of chitosan when it is functionalized with different phytochemi­ cals bearing catechol groups (Amato et al., 2018; Kim et al., 2017; Qin & Li, 2020) Therefore, in this work the growth inhibition capacity of CSRA conjugates was evaluated and compared to unmodified chitosan and free RA Notably, since bacterial concentrations were determined by optical density at 600 nm, free RA concentrations were limited by the experiment itself Concentrations higher than 3.9 μg/mL led to Credit authorship contribution statement ˜ al: Conceptualization, Methodology, Formal M Huerta-Madron analysis, Investigation, Writing – original draft, Writing – review & ´ n: Conceptualization, Methodology, editing, Visualization J Caro-Leo Formal analysis, Investigation, Writing – review & editing, M Huerta-Madro˜ nal et al Carbohydrate Polymers 273 (2021) 118619 A C B D E F 120 HFB HEK RAW G ROS production (%) 100 80 60 40 20 * * * * * * * * * Negative control Positive control CS-0.8RA CS-0.4RA Fig Photo-protective capacity of CSRA h lixiviates (A–C) cell viability after 24 h, and (D–G) intracellular reactive oxygen species production of HFB, HEK and RAW 264.7 upon UVB irradiation in presence of h lixiviates from rosmarinic acid-chitosan (CSRA) films The diagrams include the mean, SD (n = 24), and the ANOVA results (*p < 0.05 statistically significant difference between untreated and either control or treated cells, and #p < 0.05 between control and CSRA treated cells (brackets)) M Huerta-Madro˜ nal et al Carbohydrate Polymers 273 (2021) 118619 Fig Growth inhibition capacity of (A) different rosmarinic acid (RA) concentrations, and (B) h lixiviates from native chitosan (CS) and rosmarinic-acid chitosan conjugates (CSRA) films on S epidermidis and (C) E coli Ampicillin and Gentamicin were used as specific antibiotics The diagrams include the mean, SD (n = 16), and the ANOVA results (*p < 0.05 statistically significant difference between negative control and samples, and #p < 0.05 between the different samples (brackets)) Visualization E Espinosa-Cano: Conceptualization, Methodology, Formal analysis, Investigation M.R Aguilar: Conceptualization, Methodology, Writing – review & editing, Supervision, Project admin­ ´zquez-Lasa: Conceptualization, istration, Funding acquisition B Va Methodology, Writing – review & editing, Supervision, Project admin­ istration, Funding acquisition References Abbas, M., Saeed, F., Anjum, F., Afzaal, M., Tufail, T., Bashir, M., … Suleria, H A R (2017) Natural polyphenols: An overview International Journal of Food Properties, 20 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effects – A review Journal of Functional Foods, 18, 820–897 https://doi.org/10.1016/j.jff.2015.06.018 Silveira Fachel, F N., Pr´ a, M., Azambuja, J., Endres, M., Bassani, V., Koester, L., & Braganhol, E (2019) Glioprotective effect of chitosan-coated rosmarinic acid nanoemulsions against lipopolysaccharide-induced inflammation and oxidative stress in rat astrocyte primary cultures Cellular and Molecular Neurobiology, 40(1), 123–139 Singh, B R (1999) Basic aspects of the technique and applications of infrared spectroscopy of peptides and proteins In Infrared Analysis of Peptides and Proteins (Vol 750, Issue 750, pp 2–37) American Chemical Society doi:doi:https://doi org/10.1021/bk-2000-0750.ch00110.1021/bk-2000-0750.ch001 12 ... to a new material with properties already described for rosmarinic acid (i.e antioxidant, anti-inflammatory, and photoprotective) and for chitosan (i.e antimicrobial activity and biodegradability)... different rosmarinic acid (RA) concentrations, and (B) h lixiviates from native chitosan (CS) and rosmarinic- acid chitosan conjugates (CSRA) films on S epidermidis and (C) E coli Ampicillin and Gentamicin... applications Materials and methods 2.1 Synthesis and characterization of rosmarinic acid -chitosan conjugates Rosmarinic acid (RA, 96% pure, Merck KGaA, Darmstadt, Germany) was conjugated to chitosan (CS,