The states of water in tryptophan grafted hydroxypropyl methylcellulose hydrogels and their effect on the adsorption of methylene blue and rhodamine B

Tryptophan (Trp) decorated hydroxypropyl methylcellulose (HPMC) cryogels were prepared by a one-step reaction with citric acid. The increase of Trp content in the 3D network from 0 to 2.18 wt% increased the apparent density from 0.0267 g.cm−3 to 0.0381 g.cm−3 and the compression modulus from 94 kPa to 201 kPa, due to hydrophobic interactions between Trp molecules.

Carbohydrate Polymers 248 (2020) 116765 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol The states of water in tryptophan grafted hydroxypropyl methylcellulose hydrogels and their effect on the adsorption of methylene blue and rhodamine B T Paulo V.O Toledoa, Oigres D Bernardinellib, Edvaldo Sabadinib, Denise F.S Petria,* a b Fundamental Chemistry Department, Institute of Chemistry, University of São Paulo, Av Prof Lineu Prestes 748, 05508-000, São Paulo, Brazil Department of Physicochemistry, Institute of Chemistry, University of Campinas (UNICAMP), 13083-970, Campinas, São Paulo, Brazil A R T I C LE I N FO A B S T R A C T Chemical compounds studied in the article: Hydroxypropyl methylcellulose Citric acid Tryptophan Methylene blue Rhodamine B Tryptophan (Trp) decorated hydroxypropyl methylcellulose (HPMC) cryogels were prepared by a one-step reaction with citric acid The increase of Trp content in the 3D network from to 2.18 wt% increased the apparent density from 0.0267 g.cm−3 to 0.0381 g.cm−3 and the compression modulus from 94 kPa to 201 kPa, due to hydrophobic interactions between Trp molecules The increase of Trp content in HPMC-Trp hydrogels increased the amount of non-freezing water, estimated from differential scanning calorimetry, and the amount of freezing water, which was determined by time-domain nuclear magnetic resonance The adsorption capacity of methylene blue (MB) and rhodamine B (RB) on HPMC-Trp hydrogels increased with Trp content and the amount of freezing water HPMC-Trp hydrogels could be recycled times keeping the original adsorptive capacity The diffusional constants of MB and RB tended to increase with Trp content RB adsorbed on HPMC-Trp hydrogels presented a bathochromic shift of fluorescence Keywords: Hydroxypropyl methylcellulose Tryptophan States of water Hydrogels Cryogels Adsorption Introduction Polysaccharide based 3D structures, such as aerogels and cryogels, are interesting platforms for tissue engineering (Tchobanian, Van Oosterwyck, & Fardim, 2019), drug delivery (Ulker & Erkey, 2014) and adsorption of pollutants (Maleki, 2016) Upon contact with aqueous media, the aerogels or cryogels become hydrogels Understanding the structure of water around the polysaccharide chains is important because it might drive the interactions with cells, drugs or pollutants Water molecules in hydrogels coexist as three different states: (i) Nonfreezing bound water or non-freezing water (Wnf), which results from tightly bound water molecules, it is not freezable at °C or below °C, due to the strong interaction with the polymer chain (ii) freezing bound water or intermediate water (Wfb), which is not freezable at °C, but it is freezable below °C because the interactions with polymer chains are not so strong as in the Wnf, and (iii) free water or freezing water (Wf), which is freezable at °C, in this case, water molecules hardly interact with the polymer chains, they are just entrapped in the matrix (Tsuruta, 2010) The amount of water molecules in each state depends fundamentally on the chemical nature of polymer (Hatakeyama & Hatakeyama, 2017) Hydroxypropyl methylcellulose (HPMC) is a family of water-soluble cellulose ethers widely applied in cosmetics (Lochhead, 2017), pharmaceuticals (Kaur et al., 2018), and food (Burdock, 2007) formulation HPMC chains can be crosslinked via esterification with citric acid (CA), a nontoxic multifunctional acid, enabling the formation of tridimensional structures for drug release (Marani, Bloisi, & Petri, 2015; Reddy & Yang, 2010) or adsorption of pollutants (Martins, Toledo, & Petri, 2017; Toledo et al., 2019) Cellulose can be modified with amino acids in order to improve bioaffinity (Kalaskar et al., 2008) or to develop membrane for methanol fuel cells (Zhao et al., 2019) Synthetic polymers modified with L-tryptophan (Trp), a hydrophobic essential amino acid (Richard et al., 2009), can be used to produce cryogels with high affinity for proteins (Türkmen et al., 2015) or DNA (Çorman, Bereli, Ưzkara, Uzun, & Denizli, 2013) Despite the above-mentioned potential applications, systematic investigations about cryogels and hydrogels based on amino acid modified HPMC, the consequences on the mechanical properties, on the states of water in such materials and on their ⁎ Corresponding author E-mail addresses: paulo.vinicius.toledo@usp.br (P.V.O Toledo), oigres.daniel@gmail.com (O.D Bernardinelli), sabadini@unicamp.br (E Sabadini), dfsp@iq.usp.br (D.F.S Petri) https://doi.org/10.1016/j.carbpol.2020.116765 Received 20 February 2020; Received in revised form 27 June 2020; Accepted 11 July 2020 Available online 25 July 2020 0144-8617/ © 2020 Elsevier Ltd All rights reserved Carbohydrate Polymers 248 (2020) 116765 P.V.O Toledo, et al Fig Schematic representation of (a) HPMC, (b) CA, (c) Trp, (d) MB and (e) RB Experimental Table CA:Trp molar ratios used in the precursor gels to synthesize the HPMC-Trp cryogels Nitrogen content determined by elemental analyses (N, %), calculated Trp content (wt%) and gel content (%) CA:Trp N (%) Trp content (wt%) Gel content (%) 1.0:0.0 1.0:0.5 1.0:1.0 2.0:0.5 2.0:1.0 2.0:2.0 0.10 ± 0.01 0.15 ± 0.01 0.16 ± 0.01 0.19 ± 0.01 0.31 ± 0.01 0.73 ± 0.07 1.09 ± 0.07 1.17 ± 0.07 1.38 ± 0.07 2.18 ± 0.07 93 ± 89 ± 82 ± 89 ± 85 ± 80.5 ± 0.8 2.1 Preparation of Tryptophan decorated cryogels HPMC E4M (USP HPMC 2910, MS 0.25, DS 1.9) was kindly provided by The Dow Chemical Company (Brazil), GPC measurements (Supplementary Material SM1) indicated Mn 1.1x105 g mol−1 and Mw 2.4x105 g mol−1 The crosslinking of HPMC chains and the attachment of Trp to the HPMC chains were performed with CA in a onestep reaction Briefly, aqueous solutions containing HPMC at 2.0 wt%, CA at 0.10 wt% or 0.20 wt% (CA, LabSynth, Brazil, 192.13 g.mol−1), sodium hypophosphite monohydrate at 0.05 wt% (HPS, LabSynth, Brazil, 106.14 g.mol−1) and L-tryptophan (Trp, Sigma-Aldrich, 204.23 g.mol−1, T0254) were stored in the refrigerator at 12 °C for h in order to achieve complete dissolution of HPMC The solutions were prepared at different CA:Trp molar ratios of 1.0:0.0, 1.0:0.5, 1.0:1.0, 2.0:0.5, 2.0:1.0, and 2.0:2.0 The highest concentration of Trp in solution was 2.0 g.L−1, which is well below its solubility at 25 °C of 11.4 g.L−1 [National Center for Biotechnology Information PubChem Database Tryptophan, CID = 6305, https://pubchem.ncbi.nlm.nih.gov/ compound/Tryptophan (accessed on Feb 20, 2020)] The hydrogels were poured into molds of different sizes, were frozen in a freezer (-40 °C for h) and freeze-dried (-50 °C, 200 μmHg, for 8–24 hours) The resulting 3D solid structures were heated at 165 °C for in order to promote the reaction between HPMC hydroxyl groups and CA carboxylic acid groups and/or with Trp carboxylic acid/amine groups One should notice that in the absence of CA there is no chemical crosslinking among the HPMC chains If CA mass is less than % of polymer mass, the crosslinking efficiency is very low and the hydrogels show no mechanical stability CA mass between 5% and 10 % of polymer mass yields stable HPMC hydrogels (Marani et al., 2015; Martins et al., 2017) Ghorpade and co-workers showed that CA mass of 15 % or 20 % of polysaccharide (carboxymethyl cellulose and tamarind gum) mass is not adequate because the swelling degree of hydrogels tends to decrease (Mali, Dhawale, Dias, Dhane, & Ghorpade, 2018) For compressive strength tests, rectangular samples of 13.0 mm x 17.0 mm x 25.0 mm were prepared, whereas for the other analyses the samples were discs of mm thickness and 35 mm of diameter The samples were coded as HPMC-Trp cryogels affinity for dyes, are poorly explored Dyes are environmental pollutants, thus it is important to comprehend the correlation between the states of water in hydrogels and their adsorption capacity Preliminary, HPMC cryogels were prepared with four different amino acids (AA), namely, tryptophan (Trp, hydrophobic), glutamic acid (Glu, acid), cysteine (Cys, polar) and histidine (His, basic) Among all AA modified HPMC cryogels, the HPMC-Trp cryogels presented the highest compression modulus in comparison to pure HPMC cryogels Similarly, the presence of hydrophobic particles, such as lignin, in cryogels and hydrogels improved their mechanical properties and adsorptive capacity for dyes (Zhang et al., 2019) Based on this, we proceed with the systematic investigation on the (i) crosslinking of HPMC chains and chemical attachment of Trp moieties to HPMC by esterification in one-step reaction, with different CA:Trp ratios, (ii) the physicochemical properties of HPMC-Trp cryogels, (iii) the states of water (Wnf and Wfb) in HPMC-Trp hydrogels and (iv) how the Trp content and states of water affect the adsorption capacity of HPMC-Trp hydrogels for methylene blue (MB) and rhodamine B (RB) To the best of our knowledge, it is the first systematic study about the correlation between the states of water and the adsorption behavior of dyes involving AA modified polysaccharides Carbohydrate Polymers 248 (2020) 116765 P.V.O Toledo, et al Fig (a) Dehydration of citric acid and anhydride formation upon heating (b) Esterification and crosslinking between two HPMC chains (c) Esterification of anhydride and HPMC hydroxyl group followed by attachment of Trp amino group to form an amide linkage and HPMC-Trp 2.18 wt% were compressed and decompressed in MilliQ water up to times; in the first cycle of compression/decompression, the materials presented larger hysteresis and larger mechanical resistance than in the following cycles due to the presence of air entrapped inside the cryogels, which after the first cycle was expelled by water Fourier transform infrared spectroscopy analyses in the attenuated total reflectance mode (FTIR-ATR) were performed in a Perkin Elmer Frontier equipped with ZnSe crystal, resolution of cm-1 and in the wavenumber range of 600 cm−1 to 4000 cm−1 Elemental analysis (CHN) was performed with a Perkin Elmer 2400 Series II equipment For the differential scanning calorimetry (DSC TA Instruments Q10), the samples were swollen in MilliQ water (≈ μS cm−1) at 20 °C for 12 h, degassed under vacuum pump (10 at 100 mmHg) and subject to cycles from – 40 °C to 40 °C, at °C.min−1 rate Measurements of TDNMR were performed in a Minispec 20 MHz at 33 °C T2 relaxation time measurement was carried out with a standard Carr-PurcellMeiboom-Grill (CPMG) pulse sequence with 30000 echoes and an echo time of 16 μs (Carr & Purcell, 1954) SKL Neo MultiExp program for inverse Laplace transform (ILT) was used and Log-Normal distribution integration aided by an user-guided program called Peakfit 4.00 (Jandel scientific software); the dried cryogels (≈ 25 mg) were swollen in MilliQ water (1.0 mL) at 20 °C inside the NMR probe and the results were acquired every from the completely dry sample to the absolute swelling that lasted a total of 45 2.2 HPMC-Trp cryogels characterization For the characterization, all cryogels were rinsed with MilliQ water until the rinsing water achieved conductivity of ≈ μS cm−1 This procedure removed the unreacted molecules, which could be only physically attached to the samples After that, they were freeze-dried and weighed again The gel content (Gel %) was calculated according to Eq (1): mpol − mdried ⎞ ⎤ ⎡ Gel (%) = ⎢1 − ⎜⎛ ⎟ ⎥ × 100 mpol ⎝ ⎠⎦ ⎣ (1) where mpol is the initial mass of HPMC and mdried is the mass of the freeze-dried sample The swelling degree (SD) was determined with a precision tensiometer Krüss K100 at (24 ± 1) °C as the mass of sorbed MilliQ water (pH 5.5) at equilibrium divided by the mass of dried adsorbent: SD = m water mdried (2) The apparent density of HPMC-Trp cryogels was determined at (24 ± 1) oC by dividing the mass of freeze-dried cryogels by the corresponding volume, which was estimated by their dimensions The dimensions of seven different samples of the same chemical composition were measured using a pachometer; the same seven samples were weighed in an analytical balance The mean mass divided by the mean volume determined for seven samples yielded the mean apparent density (ρap) value SEM analyses were performed for gold-coated (by sputtering) samples in a Jeol Neoscope microscope JCM 5000, operating at kV voltage The compressive tests were performed for 10 cryogels (rectangular) samples using an Impac, Digital Dynamometer IP-90DI, with a 10 N load cell, at the strain rate of 0.01 s−1 and at (24 ± 1) oC and (70 ± 5) % relative air humidity The samples HPMC 2.3 Adsorption studies Prior to the adsorption experiments, all cryogels were rinsed with MilliQ water until the rinsing water achieved conductivity of ≈ μS cm−1 After that, they were freeze-dried and weighed again For the adsorption studies, methylene blue (MB, Sigma-Aldrich, 319.81 g.mol1 ) dissolved in Tris-HCl 0.05 mol.L-1 buffer at pH 7.0 (Trizma Base, Sigma-Aldrich, 121.14 g.mol-1) and rhodamine B (RB, Sigma-Aldrich, Carbohydrate Polymers 248 (2020) 116765 P.V.O Toledo, et al Fig (a) Apparent density (ρap), (b) compression modulus (ε) as a function of Trp content in the HPMC-Trp porous materials, (c) Dependence of ε on ρap, the red line corresponds to the fit ε = k ρapm, R² = 0.9051, m = 1.8195, and (d) swelling degree (SD) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article) 479.01 g.mol-1) dissolved in Tris-HCl 0.05 mol.L-1 buffer at pH 2.5 were used as model molecules Noteworthy, these pH conditions were chosen because at pH the adsorption of MB on the walls of the vials and selfassociation by π stacking was avoided At pH higher than three, the adsorption of RB molecules on the HPMC or HPMC-Trp hydrogels was too low All adsorption experiments were performed at (24 ± 1) °C and contact time of 24 h, in order to assure equilibrium conditions (Kaewprasit, Hequet, Abidi, & Gourlot, 1998) The equilibrium adsorption capacity (qe, mg g−1) of MB or RB was calculated dividing the concentration of adsorbed MB or RB by the mass of dried cryogels (m) and multiplying by the solution volume (v): qe = C0 − Ce ×v m mL) for 15 After this period, aliquots of supernatant were withdrawn, and the concentration of released RB was determined by photometry at 553 nm Then the hydrogels were immersed in the RB solution for 10 to proceed with next adsorption/desorption cycle After this period, aliquots of supernatant were withdrawn, and the concentration of remaining RB was detected Then the next desorption process was conducted In order to get insight about the interactions between MB or RB molecules and Trp moieties bound to HPMC chains, fluorescence was measured with a Shimadzu RF6000 spectrofluorometer, at (24 ± 1) °C, at 600 nm.min−1, excitation and emission bandwidth of nm, resolution of 1.0 nm and reproducibility of 0.2 nm After 48 h adsorption of MB or RB (C0 = 1.0 mg.L-1) on HPMC-Trp hydrogels, a holder positioned at 45° with respect to the optical axis suspended the swollen hydrogels in the air Samples carrying MB or RB were excited at 650 nm or 540 nm, respectively; the emission spectra were measured in the range of 670 nm–820 nm or 550 nm–700 nm, respectively Fluorescence of MB and RB solutions at 0.33 mg.L-1 in the corresponding buffers was measured in a 10 mm x 10 mm quartz cuvette All measurements were performed for at least two samples of the same composition For comparison, the intensity values of fluorescence spectra were normalized with respect to the intensity at the maximum wavelength Fig represents the chemical structures of the main chemical compounds used in the experiments (3) The concentration of adsorbed MB or RB onto the cryogels was determined as the difference between the initial concentration (C0) of MB or RB and the concentration of MB or RB in the supernatants after 24 h contact, or the equilibrium concentration (Ce) First, calibration curves of absorbance intensity as a function of MB and RB concentration were determined by means of spectrophotometry in a Beckmann Coulter DU640 spectrophotometer, respectively at 664 nm and 553 nm (Supplementary Material SM2) For the adsorption isotherms of MB onto Trp decorated HPMC cryogels (∼30 mg dry mass), the initial concentration (Ci) of MB ranged from 0.5 mg.L−1 to 5.0 mg.L−1 For the adsorption isotherms of RB onto HPMC-Trp cryogels, the initial concentration (Ci) of RB ranged from 0.5 mg.L−1 to 4.0 mg.L−1 Adsorption/desorption cycles were carried out by immersing the RB coated samples in MilliQ water (10 Carbohydrate Polymers 248 (2020) 116765 P.V.O Toledo, et al Fig SEM images for (a) pure HPMC cryogels and HPMC-Trp cryogels with (b) 0.73 wt%, (c) 1.09 wt%, (d) 1.17 wt%, (e) 1.38 wt% and (f) 2.18 wt% Trp Scale bars correspond to 50 μm Table Experimental values of ΔHendo determined for the HPMC-Trp hydrogels by DSC, the Wt , Wnf and (Wf + Wfb) values calculated with Eqs (4) and (5) Wfb fraction was determined by TD-NMR measurements (detailed in Section 3.3) Trp (wt%) ΔHendo (J/g) 0.73 ± 0.07 1.17 ± 0.07 1.38 ± 0.07 2.18 ± 0.07 267 ± 310 ± 305 ± 0.7 297 ± 301 ± Wt (%) Wnf 97.73 ± 0.00 97.18 ± 0.05 97.78 ± 0.01 97.20 ± 0.04 97.9 ± 0.3 18 ± 4.4 ± 0.7 6.5 ± 0.2 8±2 8±2 (Wf + Wfb) (%) Wfb (%) (TD-NMR) 80 ± 92.8 ± 0.6 91.2 ± 0.2 89 ± 90 ± 13.91 65.80 21.80 38.11 92.24 (%) Results and discussion HPMC and CA carry no N atom in their structures Considering the N contents determined from elemental analyses (Table 1) and the N% content in Trp of 11.75 %, the Trp content in the cryogels was calculated (Table 1) The chemical attachment of Trp to the cryogels was favored by the increase of CA and Trp concentrations in the precursor gel; the highest Trp content of 2.18 ± 0.07 wt% was achieved for the CA:Trp molar ratio of 2.0:2.0 On the other hand, the gel content (Gel %) was the smallest at the CA:Trp molar ratio of 2.0:2.0, indicating competition between HPMC hydroxyl groups and Trp amine groups for the CA carboxylic acid CA carries three carboxylic acid groups, which in the presence of HPS and under heating undergoes dehydration and anhydride formation (Peng, Yang, & Wang, 2012) (Fig 2a) The anhydride might react with HPMC hydroxyl groups, such esterification can take place with hydroxyl groups of a second HPMC chain, promoting the crosslinking between HPMC chains (Fig 2b.) The anhydride bound to an HPMC chain can also react with Trp amino groups to form amide groups (Fig 2c), decreasing the crosslinking between two HPMC chains Thus, the increase of CA:Trp ratio increases the competition between these reactions, decreasing the crosslinking (Gel %) and increasing the attachment of Trp to HPMC chains Supplementary Material SM4 provides FTIR-ATR spectra of pure HPMC and HPMC-Trp in the 4000 to 600 cm−1 range All samples 3.1 Characterization of HPMC-Trp cryogels Preliminarily, HPMC cryogels were prepared with four different amino acids, namely, tryptophan (Trp, hydrophobic), glutamic acid (Glu, acid), cysteine (Cys, polar) and histidine (His, basic) at CA:AA molar ratio of 1.0:1.0 The corresponding compression moduli (ε) values followed the sequence: HPMC-Trp > HPMC-His > HPMCCys > HPMC-Glu > pure HPMC; the data are provided as Supplementary Material SM3 The ε values followed the relative hydrophobicity of amino acids given as Trp > > Cys > His > > Glu (Wimley & White, 1996), indicating that the inclusion of hydrophobic amino acid improved the mechanical properties of HPMC cryogels Based on this experimental observation and on the lack of reports about the physicochemical properties of Trp modified HPMC cryogels, Trp was chosen to proceed with the systematic modification of HPMC cryogels The synthesis of HPMC-Trp cryogels with different CA:Trp molar ratios led to different contents of N in the HPMC cryogels, as revealed by CHN analyses (Table 1) The N% content in the samples stems exclusively from the chemical attachment of the Trp to the cryogels, since Carbohydrate Polymers 248 (2020) 116765 P.V.O Toledo, et al Fig Representation of the T2 decay-time distribution influenced by the pore size schematically indicated in the SEM image, (a) small pores and free “bulk” water, (b) large pores and free water and (c) small and large pores and free water (d) T2 decay-time distribution of the hydrogen atoms of water measured as soon as the HPMC-Trp cryogels were put in contact with water (virtually at min) and after 45 contact The Trp content in HPMC-Trp samples varied from wt% to 2.18 wt % presented the characteristic HPMC bands: in the 3500–3200 cm−1 region (OH vibrational stretching); at 2930 cm−1 and 2850 cm−1 (symmetrical and asymmetrical CH stretching); and in the 1200–850 cm−1 region (CO and CCee stretching vibrations of the glucopyranose ring) (Silverstein, Webster, Kiemle, & Bryce, 2014) The esterification between CA and HPMC was identified by the bands at ≈1730 cm−1 and 1640 cm−1, which were assigned to C]O stretching of ester and acidic forms, respectively (Bueno, Bentini, Catalani, & Petri, 2013), the bands at 1458 cm−1, 1408 cm-1, and 1322 cm−1 were assigned to CH2 scissor, symmetric axial deformation of C]O of esters and stretching of COe of carboxylate groups HMPC-Trp presented characteristic amide vibrational bands at 1642 cm−1 and 1312 cm−1, assigned to νC=O amide I and νC-N amide, respectively (Liu, Shen, Zhou, Wang, & Deng, 2016), which overlapped other characteristic bands already observed in pure HPMC cryogels, impairing the identification of Trp by these vibrational bands However, HPMC-Trp with 1.38 wt% and 2.18 wt% Trp presented two weak bands at 748 cm−1 and 707 cm−1, which are characteristic of Trp indole ring; these bands did not appear in the spectra of pure HPMC cryogels and, therefore, indicated the chemical attachment to the HPMC hydrogels Fig 3a and b shows that the apparent density (ρap) and the compression modulus (ε) increased considerably with the increase of Trp content in the HPMC-Trp cryogels, indicating that hydrophobic interaction between Trp molecules might contributed to the cell wall structuring HPMC-Trp 2.18 wt% presented ε value twofold of that determined for pure HPMC cryogels For comparison, the ε values of HPMC cryogels modified with 15 wt% of cellulose nanocrystals increased only 20 % in comparison to pure HPMC cryogels (Toledo et al., 2019), but the addition of wt% hydrophobic lignin particles to hydrophilic poly(vinyl alcohol), PVA, hydrogels increased fourfold the ε value over that of pure PVA hydrogels (Bian et al., 2018) Thus, selfassembling of hydrophobic moieties added to hydrophilic cryogels might improve the mechanical properties of cryogels The ε values increased with ρap1.82 (Fig 3c); the index value of 1.82 is typical for isotropic open-cell structures (Scotti & Dunand, 2018) Fig shows the SEM images of pure HPMC and HPMC-Trp with different Trp contents Regardless of the Trp content, all cryogels presented isotropic open cellular structure, in agreement with the index value of 1.82 The swelling degree (SD) values (Fig 3d) presented no significant dependence on the Trp content On average, the SD for water amounted to ≈ 47 g per g of cryogel This value is ≈ 10 units smaller than those found for cryogels made of negatively charged Carbohydrate Polymers 248 (2020) 116765 P.V.O Toledo, et al Fig Adsorption capacity (qe) of (a) MB at pH and (b) RB at pH 2.5, respectively, onto HPMC-Trp hydrogels (30 ± mg dried basis and 10 mL solution) as a function of the equilibrium concentration of (Ce), at (24 ± 1) oC KF values determined for MB and RB as a function of (c) Trp content and (d) freezing bound water fraction (Wfb) where msw is the mass of swollen hydrogel at equilibrium The ratio of the melting enthalpy values of free water in the hydrogel (ΔHendo) and of ice (ΔHmo = 334 J.g−1) (Bouwstra, Salomon-de Vries, & van Miltenburg, 1995) was calculated to estimate the fraction of free water The DSC curves were provided as Supplementary Material SM6 Table shows the experimental values of ΔHendo determined for the HPMC-Trp hydrogels by DSC, the Wt , Wnf and (Wf + Wfb) values calculated with Eqs (4) and (5) All HPMC-Trp hydrogels presented lower Wnf values than bare HPMC hydrogels (17.72 %), because the amount of bound water depends strongly on the polymer hydrophilicity For instance, the increase of the degree of substitution (DS) of carboxymethyl cellulose (CMC) from 0.7 to 1.8 increased the Wnf values from ≈ 75 % to ≈ 90 % (Hatakeyama & Hatakeyama, 2017) However, among the HPMC-Trp samples, the Wnf values tended to increase with the Trp content Higher contents of Trp attached to HPMC were achieved by higher CA:Trp ratios (2.0:0.5, 2.0:1.0 and 2.0:2.0), so that not only the Trp content increased but also the number of carboxylate groups For those samples, the increase of hydrophobic Trp content from 1.17 wt% to 2.18 wt% was accompanied by the increase of hydrophilic carboxylate groups stemming from CA polysaccharides, like xanthan gum (Toledo, Marques, & Petri, 2019) or carboxymethyl cellulose (Toledo, Limeira, Siqueira, & Petri, 2019), due to the absence of charges in the HPMC structure Fig Supplementary Material SM5 shows the compressive stress-strain curves measured in MilliQ water for HPMC and HPMC-Trp 2.18 wt% The samples were compressed and decompressed up to times, but for the sake of clarity, only the 2nd cycles were presented Both HPMC and HPMC-Trp 2.18 wt % hydrogels presented high resilience, small hysteresis and similar stiffness This trend is in agreement with the independence of SD values on Trp contents (Fig 3d) 3.2 Determination of non-freezing water (Wnf) in HPMC-Trp hydrogels by means of DSC HPMC-Trp cryogels become hydrogels upon immersion in water The amount of Wnf was calculated with Eq (4) (Kim, Lee, & Kim, 2004): ΔHendo ⎞ Wnf (%) = Wt − (Wf + Wfb) = Wt − ⎛⎜ × 100 o ⎟ ⎝ ΔHm ⎠ (4) where Wt is the total water in the hydrogel, the sum Wf and Wfb corresponds to the fraction of free water One should notice that by DSC it was not possible to discriminate the Wf and Wfb due to the low polymer content Wt can be determined by Eq (5): m − mdried ⎞ × 100 Wt (%) = ⎛ sw msw ⎠ ⎝ ⎜ 3.3 Investigation of water in HPMC-Trp hydrogels through TD-NMR Fig indicates a schematic representation of T2 decay-time distribution of the hydrogen atoms corresponding to the water molecules that are filling the pores of the cryogels (SEM image) and the excess of water (free water) The intensity of the signal relates to the number of ⎟ (5) Carbohydrate Polymers 248 (2020) 116765 P.V.O Toledo, et al Fig Adsorption capacity qt (mg g−1) as a function of time t (min) determined for MB at (a) 3.0 mg.L−1 and (b) 5.0 mg.L−1, and for RB at 2.0 mg.L−1 and 4.0 mg.L−1 remained practically constant, indicating that the majority of the water was incorporated into the pores of the cryogels very fast (shorter than min) The pattern of the signals for the HPMC-Trp samples differed from the one for pure HPMC hydrogels Without HPMC-Trp 0.73 wt%, the general trend was an increase of Wnf with the increase of Trp content in the HPMC-Trp hydrogels, as shown in Table The samples with the highest Trp contents (1.38 wt% and 2.18 wt%) presented the shortest T2 values and the distributions of the two populations (small and large pores) became closer The sample HPMC-Trp 2.18 wt% presented an interesting redistribution of the free water population after 45 of exposition, the band area of free water decreased 2.5 times and the band area related to water into the small pores increased 1.7 times, in comparison to the initial contact hydrogen nuclei of water molecules inside each pore population, while the rate of decay is associated with the mobility of the molecules Surface relaxation of the wetting phase is strongly dependent on the wettability environment within the pores space (Câmara et al., 2020; Schmidt-Rohr & Spiess, 1994; Vidal, Bernardinelli, Paglarini, Sabadini, & Pollonio, 2019) For this reason, water in small pores provides short relaxation time (Fig 5a), while water residing in large pores is related to longer relaxation times (Fig 5b) Fig 5c represents the expected decay for water in a matrix containing the two pores populations The longest relaxation time corresponds to the population of free “bulk” water Fig 5d shows the distribution of T2 obtained for HPMC-Trp hydrogels with Trp content ranging from wt% to 2.18 wt% The corresponding decay curves were previously treated by the ILT, as shown in the Supplementary Material SM7 For each sample, plots for water distribution were recorded as soon as water was added to the cryogel (0’), and after 45 The characteristic signal at around 2,500−3,000 ms corresponds to the free water (excess of water) present in the NMR tube and in the hydrogels Signals at a smaller time scale were attributed to Wfb The mean T2 values and the percentage of water in each population (area of the bands) were analyzed by considering the T2 distribution at 0’ and 45’, as shown in Supplementary Material Table SM1 The areas corresponding to each band were obtained through the deconvolution of plots by using Log-Normal distribution integration, representing the fraction of water free and present in the pores (Supplementary Material SM8) The signals below ms, which might be related to bound water (Wnf) (Wang et al., 2020; Wei et al., 2018), were too weak to be accurately treated with ILT and appeared only in two samples (Supplementary Material SM9) Except for HPMC-Trp 2.18 wt%, the population of free water 3.4 Adsorption of MB and RB on HPMC-Trp hydrogels Fig 6a and b shows the equilibrium adsorption capacity (qe) of MB at pH and RB at pH 2.5, respectively, onto HPMC-Trp hydrogels (30 ± mg dried basis and 10 mL solution) as a function of the equilibrium concentration of (Ce), at (24 ± 1) oC At pH 7, MB has one negative charge in the conjugated nitrogen (Flury & Wai, 2003), whereas at pH 2.5, RB is positively charged (Ramette & Sandell, 1956) (Supplementary Material SM10) The experimental qe values increased linearly with Ce values Fittings with Langmuir and Freundlich models (Supplementary Material SM11) indicated that the adsorption behavior of MB and RB fitted better the Freundlich model Moreover, desorption experiments showed no desorption of MB or RB after 24 h immersion in the respective solvents, impairing the fitting with the Langmuir model The Freundlich model is an empirical model, which Carbohydrate Polymers 248 (2020) 116765 P.V.O Toledo, et al coefficient of the adsorbate Supplementary Material SM16 provides typical dependence of qt on t0.5 for MB and RB at 5.0 mg.L−1 and 4.0 mg.L−1, respectively; data for more dilute solutions are available as In general, two different slopes were obtained by piecewise linear regression (dot lines), which correspond to fast and slow stages The change from the transport rate regime took place after approximately 10 Supplementary Material Table SM2 shows that the kintra values decreased one order of magnitude from the 1st to the 2nd adsorption stage and increased with the increase of adsorbate concentration There was a general tendency for the increase of kintra values with the increase of Trp content in the HPMC-Trp hydrogels The adsorption kinetics was also quantitatively evaluated using the pseudo-1st order and pseudo-2nd order models, as shown in the Supplementary Material Table SM3 The adsorption kinetics of MB and RB on HPMCTrp hydrogels fitted better with the pseudo-2nd order equation than with the pseudo-1st order equation because the fittings were over a whole time range (30 min) and the calculated qe values were similar to the experimental data However, the rate constants presented no general trend regarding the Trp content Supplementary Material SM17 shows the normalized fluorescence spectra obtained for RB (solution), MB (solution), HPMC-Trp hydrogels with different Trp contents after 48 h RB or MB adsorption, with the corresponding qe values The bathochromic shifts of emission maximum of nm (0 wt% and 0.73 wt% Trp) and 11 nm (1.09 wt% Trp, 1.38 wt% and 2.18 wt%) indicated specific interactions between RB and Trp molecules Trp and dyes might form complexes by van der Waals forces, leading to bathochromic shifts and quenching effects (Doose, Neuweiler, & Sauer, 2005) On the other hand, no significant shift in the emission maximum was observed for MB adsorbed on HPMC-Trp 2.18 wt% hydrogels, only the shoulder at ≈ 750 nm presented redshift to ≈ 770 nm yields parameters KF and n, the former is related to the adsorptive capacity and the latter to the surface homogeneity (Foo & Hameed, 2010) Fig 6c and d shows the KF values determined for MB and RB as a function of Trp content and freezing bound water fraction (Wfb), respectively The general trend for MB and RB was an increase of KF values with Trp content (Fig 6c) or Wfb (Fig 6d), indicating that the adsorption capacity correlated well with intermediate water fraction in hydrogels On the other hand, the dependence of KF values on Wnf showed a maximum at ≈ 8% for both adsorbates (Supplementary Material SM12), which corresponds to the highest Trp content Therefore, the increase of Trp content caused an increase of Wfb, Wnf and KF values The n values showed no dependence on Trp content (Supplementary Material SM13); for MB and RB, the n values amounted to 1.01 ± 0.05 and 0.92 ± 0.03, indicating similar chemical homogeneity of HPMC-Trp hydrogels used for MB and RB adsorption The qe value of ≈ 1.0 mg.g−1 for MB or RB on HPMC-Trp 2.18 wt% was similar to those determined for MB on tannin-immobilized cellulose hydrogel (qe = 1,1 mg/g) (Pei et al., 2017) and for RB on carbon nanospheres (qe = 1,15 mg/g) (Qu, Zhang, Xia, Cong, & Luo, 2015) The qe value of ≈ 1.0 mg.g−1 for MB or RB on HPMC-Trp 2.18 wt% was used for estimating the number of adsorbed MB or RB molecules per Trp molecules chemically attached to the HPMC hydrogels In one gram of HPMC-Trp 2.18 wt%, there are ≈ ×10-4 moles Trp, whereas in 1.0 mg of MB and RB there are ≈ ×10-6 moles MB and ≈ ×10-6 moles MB Thus, Trp moieties are in excess in comparison to the adsorbate molecules In order to achieve complete saturation of Trp adsorbing sites with MB and RB molecules, the qe maximal values should be ∼ 33 mg.g−1 and 50 mg.g−1, respectively These values are similar to literature values determined for lignin-based adsorbents (hydrophobic surfaces) under similar pH and dye dilute range For instance, the qe maximal values for MB on organosol lignin (Zhang, Wang, Zhang, Pan, & Tao, 2016) or blends of lignin and chitosan (Albadarin et al., 2017) amounted to (∼ 20.6 mg.g−1) and (∼ 36 mg.g−1), respectively Considering dilute range of RB (> 10 mg/L), the adsorption capacity of HPMC-Trp (50 mg.g−1) was on the same order of magnitude of that determined for activated carbons obtained from lignocellulosic waste (39 mg/g) (da Silva Lacerda et al., 2015) The reusability of the Trp-HPMC hydrogels for RB was evaluated Supplementary Material SM14 shows the removal capacity of RB (Ci = 1.0 mg/L, 10 mL) by HPMC-Trp 2.18 wt% after six adsorption cycles The adsorption time was 10 min, after that the absorbance of supernatant was measured and the hydrogels were immersed in 10 mL MilliQ water for 15 For more concentrated RB solutions (< 2.0 mg/L) the complete removal was obtained after four consecutive times in contact with 10 mL of fresh MilliQ water Regardless the initial RB concentration, the hydrogels were reused six times, keeping the removal efficiency at the original level The reusability of the HPMC hydrogels for MB adsorption is also feasible by rinsing with HCl 1.0 mol/L; even after 10 recycles, the adsorption capacity was kept at original level (Toledo, Martins et al., 2019) However, HPMC-Trp hydrogels are advantageous over pure HPMC because MB molecules undergo pronounced photofading in the presence of Trp molecules (Knowles and Gurnani, 1972) Reactions involving singlet oxygen and Trp and triplet state of MB and Trp cause the photofading of MB (Smith, 1978) Supplementary Material SM15 shows that upon increasing the Trp content in the HPMC hydrogels the photofading effect becomes more evident The adsorption kinetics of MB and RB onto HPMC-Trp hydrogels was systematically investigated for MB at 3.0 mg.L−1 and 5.0 mg.L−1, and for RB at 2.0 mg.L−1 and 4.0 mg.L−1, as shown in Fig The data were fitted with the intraparticle diffusion model (Weber & Morris, 1963), which has been widely applied for the analysis of mass transfer from solution to the solid-liquid interface and the diffusion of the adsorbate into the porous media: q = kintra t 0.5 Conclusions The present study presented the chemical crosslinking and modification of HPMC chains with citric acid (CA) and Trp by one-step reaction, creating new functional polysaccharides Upon increasing the CA:Trp ratio to 2.0:2.0 in the synthesis, the Trp content in the HPMC cryogels increased to 2.18 wt%, enhancing the compression modulus from 94 ± kPa (pure HPMC cryogel) to 201 ± kPa The insertion of hydrophobic moieties increased the Wnf fraction and the adsorption capacity for RB and MB Trp-HPMC hydrogels presented resilience and reusability The fluorescence experiments evidenced specific interactions between RB and Trp, but they were absent (or very weak) in the case of MB Therefore, the adsorption capacity oh Trp-HPMC hydrogels was favored not only by π-π interaction among the aromatic rings of Trp and MB or RB, but also by the increase of Wnf portion, or by the “cage” water molecules This finding is of high relevance because it demonstrated for the first time the interdependence between the adsorption of water-soluble adsorbates (dyes, drugs, cells, DNA, etc.) and the intermediate water fraction (Wnf), which can be tuned by the degree of hydrogel modification with hydrophobic moieties (Trp) CRediT authorship contribution statement Paulo V.O Toledo: Methodology, Investigation, Data curation Oigres D Bernardinelli: Methodology, Investigation, Data curation Edvaldo Sabadini: Conceptualization, Writing - review & editing Denise F.S Petri: Conceptualization, Writing - review & editing, Supervision, Funding acquisition, Project administration Acknowledgments Authors gratefully acknowledge financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Grant 306848/2017 and 421014/2018) and São Paulo Research Foundation (6) where kintra is the diffusion rate, which is proportional to the diffusion Carbohydrate Polymers 248 (2020) 116765 P.V.O Toledo, et al (FAPESP, Grant 2018/13492-2) This study was financed in part by the Coordenaỗóo de Aperfeiỗoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 Marani, P L., Bloisi, G D., & Petri, D F S (2015) Hydroxypropylmethyl cellulose films crosslinked with citric acid for control release of nicotine Cellulose, 22, 3907–3918 Martins, B F., Toledo, P V O., & Petri, D F S (2017) Hydroxypropyl methylcellulose based aerogels: Synthesis, characterization and application as adsorbents for wastewater pollutants Carbohydrate Polymers, 155, 173–181 Pei, Y., Chu, S., Chen, Y., Li, Z., Zhao, J., Liu, S., et al (2017) Tannin-immobilized cellulose hydrogel fabricated by a homogeneous reaction as a potential adsorbent for removing cationic organic dye from 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