Immobilization of papain enzyme on a hybrid support containing zinc oxide nanoparticles and chitosan for clinical applications

A new hybrid bionanomaterial composed of zinc oxide nanoparticles (ZnO NPs) and chitosan was constructed after enzymatic immobilization of papain for biomedical applications. In this work, we report the preparation and characterization steps of this bionanomaterial and its biocompatibility in vitro.

Carbohydrate Polymers 243 (2020) 116498 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Immobilization of papain enzyme on a hybrid support containing zinc oxide nanoparticles and chitosan for clinical applications T Aurileide M.B.F Soaresa, Lizia M.O Gonỗalvesa, Ruanna D.S Ferreirab, Jefferson M de Souzac, Raul Fangueirod, Michel M.M Alvesf, Fernando A.A Carvalhoe, Anderson N Mendesb, Welter Cantanhêdea,* a Departament of Chemistry, Federal University of Piauí, Teresina, Piauí, Brazil Department of Biophysics and Physiology, Federal University of Piauí, Teresina, Piauí, Brazil c Department of Fashion Design, Federal University of Piauí, Teresina, Piauí, Brazil d Center for Textile Science and Technology, University of Minho, Guimarães, Portugal e Department of Biochemistry and Pharmacology, Federal University of Piauí, Teresina, Piauí, Brazil f Department of Veterinary Morphophysiology, Federal University of Piauí, Teresina, Piauí, Brazil b A R T I C LE I N FO A B S T R A C T Keywords: Enzyme immobilization Papain Zinc oxide Chitosan Hybrid materials Clinical application A new hybrid bionanomaterial composed of zinc oxide nanoparticles (ZnO NPs) and chitosan was constructed after enzymatic immobilization of papain for biomedical applications In this work, we report the preparation and characterization steps of this bionanomaterial and its biocompatibility in vitro The properties of the immobilized papain system were investigated by transmission electron microscopy, zeta potential, DLS, UV–vis absorption spectroscopy, FTIR spectroscopy, and X-ray diffraction The prepared bionanomaterial exhibited a nanotriangular structure with a size of 150 nm and maintained the proteolytic activity of papain In vitro analyses demonstrated that the immobilized papain system decreased the activation of phagocytic cells but did not induce toxicity Based on the results obtained, we suggest that the novel bionanomaterial has great potential in biomedical applications in diseases such as psoriasis and wounds Introduction Chitosan is a natural polysaccharide composed of D-glucosamine linked to β- (1 → 4) and N-acetyl-D-glucosamine It has attracted great attention due to its beneficial properties in wound healing processes and psoriasis (Chamcheu et al., 2018; Jung et al., 2015; Patrulea, Ostafe, Borchard, & Jordan, 2015; Wu et al., 2018a), This polysaccharide is suitable for clinical applications because of its low toxicity, biocompatibility, and biodegradability (W Chen, Yue, Jiang, Liu, & Xia, 2018; Kumar, Isloor, Kumar, Inamuddin, & Asiri, 2019; Pereira et al., 2019; Sudirman, Lai, Yan, Yeh, & Kong, 2019) Some studies have reported the application of chitosan for the construction of hybrid nanostructures, having antibacterial activity, for dermatitis and other diseases (Chamcheu et al., 2018; Jung et al., 2015; Rozman et al., 2019; Wu et al., 2018a) The versatility of chitosan favors the construction of other models of nanostructures, including the incorporation of enzymes such as papain that can serve as a model for clinical and non-clinical studies Papain is a thiol protease, composed of 212–218 amino acid residues, found in the latex of Carica papaya and has gained widespread interest for potential biomedical applications (A Homaei & Samari, 2017; Pan, Zeng, Foua, Alain, & Li, 2016; Raskovic et al., 2015) Papain has been described as an enzyme with anti-inflammatory, bactericidal, and bacteriostatic characteristics and accelerates tissue regeneration, all of which are useful for debridement and wound healing (Y.-Y Chen et al., 2017; Hellebrekers, Trimbos-Kemper, Trimbos, Emeis, & Kooistra, 2000; A A Homaei, Sajedi, Sariri, Seyfzadeh, & Stevanato, 2010; Novinec & Lenarcic, 2013) Moreover, papain has been associated with improving the process of recovery and healing of skin wounds (Figueiredo Azevedo et al., 2017), healing of venous ulcers (Nunes et al., 2019), and plays an essential role as an enzymatic debridement agent capable of removing necrotic tissue from ulcers and wounds (Ramundo & Gray, 2008) Based on the literature, papain is also of great interest in clinical studies and can be used for the synthesis ☆ Hypothesis statement relevant for polysaccharides science: Chitosan polysaccharide plays a key role in the construction of a hybrid support for immobilization of papain enzyme aiming clinical applications ⁎ Corresponding author E-mail address: welter@ufpi.edu.br (W Cantanhêde) https://doi.org/10.1016/j.carbpol.2020.116498 Received 23 February 2020; Received in revised form 20 April 2020; Accepted 20 May 2020 Available online 26 May 2020 0144-8617/ © 2020 Published by Elsevier Ltd Carbohydrate Polymers 243 (2020) 116498 A.M.B.F Soares, et al 25 mL of 1.0 mol.L−1 NaOH were added to the mixture to precipitate the chitosan on the ZnO NPs The white precipitate was washed with ultrapure water until a pH was reached and was then oven-dried for h at 100 °C and reserved for immobilization of nanoparticles Mixtures of natural and synthetic polymers are of great interest in the biomedical and pharmaceutical fields because of their superior properties compared to those of individual polymers (Dutra et al., 2017) The combination of different polymers for the construction of nanoparticles can offer biomaterials an improvement in physical-chemical characteristics such as chemical and mechanical resistance, processability, permeability, biodegradability, and biocompatibility (Nunes et al., 2019; Thai et al., 2020; Yataka, Suzuki, Iijima, & Hashizume, 2020) The demand for new bionanomaterials for clinical treatment is expensive and time consuming Therefore, studies for new hybrid nanomaterials are needed (Guo, Richardson, Kong, & Liang, 2020) In view of these aspects, the present article proposes the development of a bionanomaterial, which uses the properties of papain and chitosan Thus, ZnO nanoparticles were synthesized to support chitosan and papain During the synthesis process, we sought to verify not only papain immobilization, but also its proteolytic activity The present work also investigated the compatibility of nanoparticles through in vitro tests to evaluate cytotoxicity The immobilization of papain on a nanoparticle hybrid support may allow future applications and biological tests in clinical processes for the treatment of wound healing and psoriasis In the present study, we demonstrated that papain maintains its proteolytic activity, does not have high cytotoxicity, and does not induce macrophage activation, demonstrating a biocompatibility profile 2.4 Strategy for immobilization of papain Papain was immobilized covalently on the ZnO/chitosan support by the glutaraldehyde activation method, as described by Zang and collaborators, with some modifications (Suganthi & Rajan, 2012) Briefly, 330 mg of the ZnO/chitosan support was added to 10 mL of a 2.5 % (v/ v) glutaraldehyde solution, which remained under constant stirring for h at room temperature This material was then washed three times with ultrapure water to remove the excess glutaraldehyde Regarding the activated support, 16.5 mL of a mg.mL−1 papain solution were added and kept under agitation for h at room temperature for immobilization to occur by chemical binding The slightly yellowish precipitate was washed three times with ultrapure water and finally oven-dried at 40 °C for h 2.5 Characterizations The formation of the ZnO/chitosan support was investigated by reaction with ninhydrin For this purpose, a dispersion of mg.mL−1 in phosphate-buffered saline (PBS) (pH 7.0) was prepared, followed by mL of a solution of 3% (m/v) ninhydrin in ethanol The mixture was heated for h at 100 °C The crystallinity and the organization of the synthesized material were investigated by using X-ray diffraction with an Empyrean X-ray diffractometer (Malvern PANanalytical, UK) with Co-Kα radiation and a scanning speed of 0.026°.min−1 over a range of 10° to 90° Ultraviolet-visible (UV–vis) spectroscopy measurements were performed using a double-beam UV-6100S Allcrom™ Spectrophotometer (Mapada Instruments, Shanghai, China) in quartz cuvettes with a cm optical path The infrared region (FTIR) spectra of the samples in KBr pellets were obtained using a Spectrum 100 FTIR Spectrometer (PerkinElmer, Waltham, MA, USA) in the 4000–400 cm-1 region Transmission electron microscopy (TEM) images were obtained using a TECNAI F20 microscope (FEI Technologies Inc Oregon, USA), with an acceleration voltage of 200 kV The samples were diluted with ultrapure water and dispersed with the aid of an ultrasonic bath, and then a drop of the colloidal suspension of each material (ZnO NPs, and immobilized papain) was placed on the grid, dried at room temperature, and analyzed by TEM The zeta potential and dynamic light scattering (DLS) measurements were performed using a Nanoparticle Analyzer SZ-100 (Horiba, Ltd., Kyoto, Japan) Before taking measurements, the dispersions of the nanomaterials were prepared at a concentration of 0.04 mg.mL−1 using the KCl solution (Dinâmica, Brazil) of 1.0 × 10-3 mol.L−1 and left to stir in an ultrasonic bath for at 25 °C Solutions of HCl (Dinâmica, Brazil) and KOH (Dinâmica, Brazil) with a concentration of 0.1 mol.L−1 each were used to adjust the pH of each dispersion The zeta potential was registered as the mean value of three measurements Materials and methods 2.1 Materials Glacial acetic acid, zinc acetate II dihydrate [Zn(CH3COO)22H2O], glutaraldehyde (25 %), and sodium hydroxide were obtained from Vetec™ Quimica Fina Ltda (Duque de Caxias, Brazil), Labsynth® Products Laboratories (Vila Nogueira, Diadema, Brazil), Sigma-Aldrich (Steinheim, Germany), and Paper Impex USA Inc (Philadelphia, PA, USA), respectively The chitosan biopolymer, from PolymarCiờncia e Nutriỗóo S/A, located in the Technological Development Park at Federal University of Ceará (Padetec-UFC Brazil), was used in the preparation of × 10−3g.L-1 of the chitosan solution using 1.0 % acetic acid (pH 5.0) Papain (Carica papaya) was obtained from the compound pharmacy Galen (Teresina, Brazil) All solutions used were prepared with ultrapure water from the PURELAB® Option-Q System (Elga LabWater, Celle, Germany) with 18.2 MΩ cm resistivity All reagents used were of analytical grade 2.2 Synthesis of zinc oxide nanoparticles (ZnO NPs) ZnO NPs were synthesized by the co-precipitation method as described by Pudukudy and collaborators, with certain modifications (Pudukudy, Hetieqa, & Yaakob, 2014) NaOH (50 mL of 0.1 mol.L−1) was added drop-wise into a reaction flask containing 25 mL of zinc acetate dehydrate 0.1 mol.L-1 to form a colloidal suspension with a white color The mixture was kept under constant magnetic stirring for 90 at 25 °C After decantation, the precipitate was washed three times with ultrapure water, oven-dried at 100 °C for h, and finally calcined in a muffle furnace for h at 300 °C at a heating rate of 10 °C.min−1 2.6 Immobilized enzyme activity tests 2.6.1 Collagen hydrolysis The proteolytic capacity of the immobilized enzyme was evaluated using a gelatin test In this assay, 10 mL of gelatin (prepared following the manufacturer’s instructions) were added to three test tubes labeled as tube A, B, and C Then, in tube A, mL of water were added to the negative control; for tube B, mL of free papain were added at mg.mL−1 for the positive control, and mL of immobilized papain were added at 10 mg.mL−1 in tube C All tubes remained under constant stirring until homogenization occurred, and then were cooled for h The proteolysis capacity of the immobilized enzyme was evaluated by observing gel formation 2.3 Synthesis of the ZnO/chitosan support To prepare the ZnO/chitosan support, 125 mg of chitosan was completely dissolved in 25 mL of acetic acid (1% v/v) and transferred to a reaction flask Then, 350 mg of ZnO NPs prepared from the previous step were added to the chitosan solution and held for 30 under constant magnetic stirring to disperse the solution Subsequently, Carbohydrate Polymers 243 (2020) 116498 A.M.B.F Soares, et al of 80 μg.mL−1) After 48 h of incubation at 37 °C and 5% CO2, 10 μL of 2% neutral red DMSO solution were added, followed by incubation for 30 Then, the supernatant was discarded, and the wells were washed with 0.9 % saline at 37 °C, and 100 μL of extraction solution were added to solubilize the neutral red present inside the lysosomal secretory vesicles The evaluation took place in a spectrophotometer after 30 at 550 nm For the evaluation of the phagocytic capacity, peritoneal macrophages were plated and incubated with the test solution After 48 h incubation at 37 °C and 5% CO2, 10 μL of stained zymosan solution were added and incubated for 30 at 37 °C followed by the addition of 100 μL of Baker's fixative to paralyze the phagocytosis process After 30 min, the plate was washed with 0.9 % saline to remove zymosan and the neutral red that was not phagocytosed by macrophages Finally, the supernatant was removed, 100 μL of extraction solution were added and the solution was analyzed using a spectrophotometer at 550 nm 2.6.2 Degradation of casein by immobilized papain To evaluate the degradability of casein after the immobilization of papain, a mg.mL−1 dispersion of immobilized papain system was initially prepared and transferred to a reaction flask containing 10 mL of bovine milk The mixture was then stirred constantly for 24 h at room temperature Casein degradation was accompanied by a UV–vis spectrophotometer, and the spectra were recorded from a dispersion containing one drop of the reaction medium in mL of water without reaction and after 24 h (Albanell et al., 2003; Luginbühl, 2002; Webster, 1970) 2.7 Animals and protocols The cells utilized were macrophages from BALB/c mice The macrophages were isolated from BALB/c mice (males or females, 5–6-weekold (20–25 g)) from Núcleo de Pesquisa em Plantas Medicinais (NPPM/ CCS/UFPI) All the experiments were performed with the authorization (no 022/15) of the Ethics Committee on Animal Experimentation, Federal University of Piauí 2.9 Statistical analysis All assays in vitro were performed in triplicates in three independent experiments; data are expressed as mean ± SEM Analysis of variance (ANOVA) followed by a Dunnet's test were performed, taking a *p < 0.05; **p < 0.01, ∗∗∗P < 0.001 required for statistical significance 2.8 Cytotoxicity evaluation: Hemolysis, MTT assay in macrophages, induction of nitric oxide synthesis, lysosomal activity, and phagocytic capacity The hemolysis test was performed on chitosan and its derivatives, according to Mendes et al (2017), with adaptations Samples of free and immobilized papain were diluted in a saline solution at the concentrations of 12.5, 25, 50, 100, 200, 400, and 800 μg.mL−1 Erythrocyte arterial blood (Ovis aries) was incubated for h at 37 °C with different samples The samples were centrifuged at 1610 × g for The supernatant was transferred to a 96-well plate and quantified at 550 nm using an EL800 Plate Spectrophotometer (BioTek Instruments, Winooski, VT, USA) In 96-well plates, 100 μL of Roswell Park Memorial Institute (RPMI) 1640 culture medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with × 105 macrophages per well were added and incubated at 37 °C with 5% CO2 for h to allow for cell adhesion Two washes with RPMI 1640 were performed to remove the cells that did not adhere Then, 100 μL of RPMI medium with the free or immobilized enzyme diluted to the concentrations of 800 to 6.25 μg.mL−1 were added, in triplicates The wells were incubated for 48 h Then, 10 μL of diluted 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were added to RPMI 1640 culture media (5 mg.mL−1) and incubated at 37 °C and 5% CO2 After discarding the medium, 100 μL of dimethyl sulfoxide (DMSO) were added to all wells The plate was shaken for 30 for the complete dissolution of MTT-formazan, and then the absorbance at 550 nm was monitored on a microplate reader, and the results were expressed in percentages The negative control was prepared with RPMI medium in 0.2 % DMSO To evaluate the activity of induction of nitric oxide synthesis, macrophages were plated in 96-well plates, with approximately × 105 macrophages per well, with solutions of pure papain and immobilized papain at concentrations of 800 to 6.25 μg.mL−1, in triplicates at 37 °C and 5% CO2 Cell culture supernatants were transferred to 96-well plates to determine the nitrite concentration The standard curve was prepared with sodium nitrite in Milli-Q® water at the concentrations of 1, 5, 10, 25, 50, 75, 100, and 150 μM diluted in RPMI 1640 medium For the nitrite dosage, equal parts of the prepared solutions (50 μL each) were used to obtain the standard curve with the same volume of the Griess reagent (1% Sulfanilamide in 10 % H3PO4 (v:v) in Milli-Q® water, added in portions equal to 0.1 % naphthylenediamine in Milli-Q® water) and the absorbance was read in a plate reader at 550 nm, with the result plotted as a percentage (Tumer et al., 2007) For lysosomal activity, peritoneal macrophages were plated and incubated with pure papain and immobilized papain (at a concentration Results and discussion 3.1 Synthesis, molecular structure, and crystallinity Fig illustrates the synthesis steps of the immobilized papain system Papain was immobilized on substrates containing ZnO NPs and chitosan by the glutaraldehyde activation method Initially, the chitosan was dissolved in acetic acid (pH 5.0), resulting in the protonation of -NH2 groups present in the structure of the biopolymer, and then ZnO NPs were added for the formation of the ZnO/chitosan support The resulting electrostatic interactions and hydrogen bonds occurred because of the interaction between the oxygen atoms of the NPs with the nitrogen and oxygen atoms of chitosan The -NH2 groups of chitosan were converted to aldehyde groups via the addition of glutaraldehyde, where the enzyme is covalently attached to the support (Fahami & Beall, 2015; Qi & Xu, 2004) The organization and molecular structure of the synthesized materials and their precursors were investigated by X-ray diffraction (XRD) Figure S1a (Supporting Information) shows that the peaks at 2θ = 12° and 2θ = 23° are attributed to the crystalline network of chitosan The strong intermolecular and intramolecular interactions between the hydrogen bonds existing in the hydroxyl, amine, and other functional groups provided a semicrystalline profile to this polymer (Krishnamoorthy, Manivannan, Kim, Jeyasubramanian, & Premanathan, 2012; Vishu Kumar, Varadaraj, Lalitha, & Tharanathan, 2004) The diffraction peak at 2θ = 23° present in the papain diffractogram (Figure S1b) indicates the large extent of the active sites of the enzyme that can be divided into domains, each of which accommodates an amino acid residue of the peptide substrate In these domains, there is a substrate-specificity space where substrate fitting occurs, which results in a semicrystalline profile for papain (Schechter & Berger, 1967; Schroder, Phillips, Garman, Harlos, & Crawford, 1993) As shown in Figure S1 (c), the ZnO NPs exhibited 10 crystalline peaks at 2θ = 37°, 40°, 32°, 55°, 66°, 74°, 79°, 81°, 82°, and 87°, which are the 100, 002, 101, 102, 110, 103, 200, 112, 201, and 202 planes, respectively, corresponding to the hexagonal wurtzite-like structure, according to the JCPDS standard No 03-065-3411 In the crystallographic pattern of the ZnO/chitosan support, the presence of all the crystalline peaks related to the hexagonal phase of the ZnO NPs (Figure S1d) was observed, along with the presence of a low-intensity peak at Carbohydrate Polymers 243 (2020) 116498 A.M.B.F Soares, et al Fig Schematic representation for the fabrication of immobilized papain system 3.2 Spectroscopic investigation, reactivity, and supramolecular arrangement 2θ = 23° (Vishu Kumar et al., 2004) The chitosan crystalline lattice, which is present in the ZnO/chitosan support, had a certain degree of crystallinity As shown in the diffractogram of the immobilized papain (Fig 2a), the peak at 2θ = 23° most likely occurs because of an overlap of chitosan and papain peaks, which also have a degree of crystallinity However, the characteristic of the peak suggests that it comes from papain (Schechter & Berger, 1967) Another factor that can be observed by the XRD technique is the crystallinity index of the materials, which was calculated according to Eq 1, where Xc is the crystallinity index, Ic is the sum of the integrals of the peak areas, and Ia is the area of the peaks of the amorphous fraction For this calculation, we considered the ranges of 10° to 30° and 30° to 90° as the amorphous and the crystalline fraction, respectively Table shows the crystallinity indices for ZnO NPs, ZnO/chitosan support substrate, and the enzyme immobilized on the ZnO/chitosan support Xc = Ic * 100 (Ic + Ia) Molecular absorption spectroscopy analysis in the visible, ultraviolet (UV) region (UV–vis) was performed to investigate the formation of the materials in colloidal suspensions The formation of nanoparticulate materials was evidenced by the increase in the baseline, which was caused by light scattering Thus, according to Mie’s law (Eq 2), when irradiating a beam of light under a colloidal suspension, the total absorption or extinction coefficient (αext) is equal to the sum of the absorbed radiation (αabs) and the scattered radiation (αsct) (Carvalho et al., 2015), as observed in the absorption spectra of ZnO NPs α ext = (αabs ) + αsct (2) Figure S2a shows the absorption spectra in the UV–vis region for free papain and ZnO NPs The absorption at 192 nm in the papain spectrum was attributed to the electron transition from the nonbinding orbital n to the anti-bonding unoccupied orbital σ (n → σ*) This type of transition is possibly due to the presence of amine and amide groups present in the enzyme structure (Feng, Zhang, Xu, & Wang, 2013; Ramimoghadam, Bin Hussein, & Taufiq-Yap, 2013; Zak, Razali, Majid, & Darroudi, 2011) For the ZnO NPs, the absorption band at 371 nm was attributed to the ZnO intrinsic bandgap absorption, characteristic of this semiconductor material, due to electron transitions from the valence band to the conduction band (O2p → Zn3d) (Azarang, Shuhaimi, Yousefi, Moradi Golsheikh, & Sookhakian, 2014) In the UV–vis spectra of ZnO/chitosan (Figure S2b), the absorption at 192 nm was attributed to the transition (n → σ*) from the -NH2 groups present in the chitosan structure Absorption at 362 nm was also observed, which was attributed to the intrinsic band gap transition of the ZnO NPs present in the ZnO/chitosan support The materials caused an increase in the baseline due to light scattering, a characteristic of nanometric-scale materials (Melo, Luz, Iost, Nantes, & Crespilho, 2013) The presence of chitosan in the support has also been qualitatively demonstrated through the ninhydrin reaction (1) It can be observed from the diffractograms that the crystalline peaks of the ZnO NPs have slightly lower intensities than the peaks of the ZnO/chitosan support This suggests an increase in the degree of crystallinity with the addition of chitosan to the ZnO NPs, which was confirmed by the value of Xc A similar behavior was observed for the immobilized enzyme In addition, the narrowing of the peak at approximately 23° confers a higher degree of crystallinity to the immobilized enzyme in ZnO NPs The gradual increase of the crystallinity index by the addition of chitosan and papain to ZnO NPs suggests that the organization of these materials is present in the structure of ZnO NPs Intriguingly, in the diffractograms of the ZnO/chitosan and immobilized papain, there was a change in the peak corresponding to the plane 002, indicating that the interaction between these materials occurs through the surface of the ZnO NPs unit cell It is known that the crystallinity index of a material is related to its size Carbohydrate Polymers 243 (2020) 116498 A.M.B.F Soares, et al Fig FTIR spectra for (a) Chitosan, (b) Free papain, (c) ZnO NPs, (d) ZnO/ Chitosan, (e) ZnO/Chitosan-glutaraldehyde and (f) immobilized papain 1583, 1418, and 1315 cm−1 were related to amide I, ™N-H deformation present in the -NH2 group, axial deformation υC-N, and amide III, respectively The vibrations at 1074 and 1034 cm−1 were attributed to the CO stretches of C3−OH and C6e−OH (Cai et al., 2015; Moradi Dehaghi, Rahmanifar, Moradi, & Azar, 2014) For free papain, the broad peak between 3594 and 3033 cm-1 and the peak at 2931 cm−1 were attributed to the OH, υN-H of the secondary amine, and υassC-H (sp3), respectively (Fig 3b) The FTIR spectrum of papain also exhibited stretches at 1638 and 1532 cm−1, corresponding to υC = O stretching vibrations of amide I and II, respectively, in agreement with published results (Mahmoud, Lam, Hrapovic, & Luong, 2013) In the region between 1074 and 928 cm−1, an overlap of several bands was observed, in which the deformations were present at 1050 cm−1, 1076 cm−1, and 844 cm−1 from the C–S stretches of sulfide and disulfide [132] For ZnO NPs, vibration at 3646 cm−1 was attributed to υO-H due to the presence of hydration water (Fig 3c) The stretches at 1642 cm−1 and 1448 cm−1 are attributed to the asymmetric and symmetrical C]O vibrations, respectively, belonging to the acetate group (not removed during washings) (Sharma, Sharma, Panda, & Majumdar, 2011) However, the increase in the calcination temperature of the ZnO NPs leads to the formation of NPs with properties similar to those of the ZnO NPs, both modified and of a larger size (Pudukudy et al., 2014; Sharma et al., 2011) The intense deformation at 497 cm−1 in the spectrum was attributed to the υZn-O deformation It can be observed that for ZnO/chitosan (Fig 3d), there were stretches and deformations from pure chitosan as well as a new deformation at 497 cm−1 that was attributed to Zn-O vibration due to electrostatic interactions and the formation of hydrogen bonds and ZnO NPS and chitosan (Cai et al., 2015; Graham et al., 2018) The immobilization of papain in the proposed substrate was confirmed by FTIR Fig 3e shows the increase in absorption at 1660 cm−1 for ZnO/ chitosan-glutaraldehyde and ZnO/chitosan-papain and at 1664 cm−1, (Fig 3f), confirming the formation of the N]C bond between the ZnO/ chitosan support and the enzyme The formation of the N]C bond is possible because of the presence of the -NH2 groups on the support and the papain, since the nucleophilic amine groups attack the aldehyde groups present in the glutaraldehyde forming an imine bond, as observed by FTIR (Hanefeld, Gardossi, & Magner, 2009; Krajewska, 2004) The supramolecular organization, morphology, and size of the ZnO NPs and immobilized papain were analyzed by transmission electron microscopy (TEM) The ZnO NPs (Fig 4a) presented a polydispersed organization with triangular shapes (nanotriangles) and the presence of Fig a) X-ray diffraction (XRD) for Immobilized Papain and b) UV–vis spectrum for reaction ninhydrin with ZnO/Chitosan (I: Before and II: After) Table Index of crystallinity of the synthesized materials Material Index of crystallinity (%) Xc ZnO NPs ZnO/chitosan Immobilized papain 81.18 83.47 86.83 Under certain conditions, ninhydrin reacts with free -NH2 groups to form a purple-colored compound, diketohydrindylidene-diketohydrindamine, known as Purple of Ruhemann This reaction can be confirmed by UV–vis, since Ruhemann’s purple exhibits a characteristic absorption at around 568 nm, as shown in Fig 2b (Lu, 2013) The inset in Fig 2b shows the color change of the solution before and after the reaction with ninhydrin In the electron spectrum in the UV–vis region for the immobilized enzyme, shown in Figure S2c, it was observed that the characteristic absorption of ZnO NPs shifted to 379 nm, which may be indicative of intermolecular interactions The formation of the molecular structure of the nanomaterials was also investigated by FTIR Fig shows the FTIR spectra of pure chitosan, free papain, ZnO NPs, ZnO/chitosan, and immobilized papain For chitosan, the peak at around 3350 cm−1 was attributed to the OH and NHee stretches because of the intra- and intermolecular hydrogen bonds of the chitosan molecules (Fig 3a) The stretches at 2931 and 2875 cm−1 are typical of υC-H, while the strain vibrations at 1658, Carbohydrate Polymers 243 (2020) 116498 A.M.B.F Soares, et al Fig Supramolecular arrangement and particle size Transmission Electron Microscopy (TEM) images: (a) ZnO NPs and (b) immobilized papain with scale of 500 nm Particle size distribution histograms for (c) ZnO NPs and (d) immobilized papain explaining why the nanoparticles did not conglomerate (Wu et al., 2018b) The presence of the chitosan biopolymer induced a decrease in the zeta potential of the ZnO nanoparticles from − 31.2 to − 25.4 This could be due to the density of the positive charge provided by chitosan and its large molecular size After papain immobilization, no change in the zeta potential was observed However, ZnO particles are unstable at pH values below 7.0, due to the formation of Zn2+ and ZnO22− species, respectively, as observed in the Pourbaix diagram (Al-Hinai, Al-Hinai, & Dutta, 2014) At pH 7.0, isolated ZnO NPs exhibited a hydrodynamic diameter equal to 612.2 ± 96 nm, which decreased after the incorporation of chitosan (504.1 ± 35.6), most likely due to the preparation of ZnO/ chitosan, since the dispersion of ZnO NPs must be carried out in acetic acid solution (pH 5.0) because of the low solubility of chitosan at a higher pH This leads to a decrease in the concentration of ZnO and corrosion of the surface of the particles (Al-Hinai, Al-Hinai, & Dutta, 2014) The hydrodynamic diameter of the immobilized enzyme demonstrates that the hydrodynamic thickness of the layer decreased from 504.1 ± 35.6–395.3 ± 91.3 during the cross-linking reaction and binding of the enzyme, which is most likely associated with the higher solvation power of chitosan in comparison to the system that contains aggregates (Salavati-Niasari, Mir, & Davar, 2009) The particle size distribution histogram of the nanotriangles (n = 113 particles) revealed an average diameter of 193 nm, with a prevalence of NPs with a size of approximately 200 nm (Fig 4b) The triangular shape of the ZnO NPs can be explained by the ability of the solvent to stabilize the water on the crystalline crystal growth planes (Salavati-Niasari et al., 2009) The immobilized enzyme system (Fig 4c) showed a supramolecular organization similar to that of the ZnO NPs The mean size of the nanotriangles of the immobilized papain estimated with 223 particles was 153 nm (Fig 4d) It is important to highlight that incorporation of chitosan, even cross-linking reaction, and the binding of the enzyme, does not change the shape of ZnO, as observed by Kumar et al (2019) and Rodrigues et al (2018) In order to understand the decrease in the size of the nanostructure after incorporating layers of chitosan and the enzyme as well as estimate the surface charge, suspension stability, and surface interaction, dynamic light scattering (DLS) and zeta-potential were performed Zeta potentials and hydrodynamic diameters of ZnO particles in the presence and absence of chitosan as well as after enzymatic immobilization in an aqueous medium (pH 7) are reported in Table S1 The zeta potential values for ZnO and derived systems showed excellent colloidal stability (Regiel-Futyra, Kus-Liśkiewicz, Wojtyła, Stochel, & Macyk, 2015), Carbohydrate Polymers 243 (2020) 116498 A.M.B.F Soares, et al papain on the surface, increasing its hydrodynamic diameter In addition, the activation of ZnO/chitosan with glutaraldehyde functionalizes the surface with aldehyde groups and condenses the chitosan molecules in ZnO NPs (Zhang et al., 2012), thus decreasing their hydrodynamic diameter These findings are in agreement with the TEM images results, in which the decrease in ZnO nanoparticle size after chitosan and enzyme immobilization was observed 3.3 Proteolytic activity of the immobilized papain Evaluation of the proteolytic capacity of immobilized papain is necessary because the immobilization of enzymes via covalent binding can worsen the catalytic performance of the protein (Krajewska, 2004) Figure S3 shows the results of the qualitative test performed using commercial gelatin to assess the proteolytic activity of immobilized papain In this assay, it was observed that for the negative control (Figure S3a), there was total gelation, in which there was no hydrolysis of the protein present in the gelatin since there was no proteolytic enzyme in the medium Figures S3b (free papain) and S3c (immobilized papain) show proteolytic activity resulting in non-gel formation due to the presence of papain in the medium causing the hydrolysis of the protein molecules present in the gelatin, disrupting the gelation process The casein hydrolysis reaction is a methodology used to evaluate the proteolytic activity of immobilized enzymes It is known that bovine milk has a high content of casein and is therefore an accessible material to acquire protein (Fahami & Beall, 2015) Fig shows the absorption spectrum in the UV–vis region for a milk sample diluted in water The presence of absorption at 276 nm was attributed to casein molecules and aromatic amino acids present in milk (Fahami & Beall, 2015) The casein hydrolysis reaction by immobilized papain was accompanied by spectrophotometric results in the UV–vis region It is interesting to note that papain can hydrolyze the casein present in milk to smaller peptides (Fahami & Beall, 2015), allowing for the evaluation of its proteolytic activity by this technique The characteristic absorption of casein decreased over time until it disappeared entirely within 24 h (Fig 5), showing that the immobilized enzyme did not lose its activity 3.4 Cytotoxicity evaluation: Hemolysis, MTT assay in macrophages, induction of nitric oxide synthesis, lysosomal activity, and phagocytic capacity Fig shows the activity of free and immobilized papain in the cell Fig Cell toxicity Hemolytic activity: (a) free papain and (b) Immobilized papain; macrophages periteneous murines cytotoxic effect: (c) free papain and (d) Immobilized papain Data are presented as mean ± SEM, obtained from three independent experiments (n = 3) in triplicate ANOVA: Dunnet's test, *p < 0.05; **p < 0.01, ∗∗∗P < 0.001 toxicity tests It was verified that the immobilized papain did not induce hemolytic activity (Fig 6a and b) at the test concentrations Fig 6c shows the cytotoxicity evaluation of free papain in murine peritoneal macrophages using the MTT assay It was observed that the free enzyme had low cytotoxicity at the studied concentration, and the CC50 for the Fig Electron spectra in the UV–vis region for pure milk and proteolytic activity test for immobilized papain after 24 h Inset: spectra absorbance zoom of 190 – 315 nm Carbohydrate Polymers 243 (2020) 116498 A.M.B.F Soares, et al Fig Evaluation of macrophage cell activation Evaluation of phagocytic activity by the production of nitrite (indirect production of nitric oxide): (a) free papain and (b) Immobilized papain; evaluation of lysossomal volume: (c) free papain and (d) Immobilized papain; evaluation of phagocytic capacity: (e) free papain and (f) Immobilized papain Data are presented as mean ± SEM, obtained from three independent experiments (n = 3) in triplicate ANOVA: Dunnet's test, *p < 0.05; **p < 0.01, ∗∗∗P < 0.001 dead cells, debris, tumor cells, and foreign materials (Hirayama, Iida, & Nakase, 2017) One way to investigate the activity of macrophages is through phagocytic activity and nitric oxide dosage, a product produced during the process of phagolysosome formation (Cape & Hurst, 2009; Tumer et al., 2007) Thus, nitrite production activity was evaluated to verify the behavior of macrophages when stimulated with ZnO NPs (Fig 7a and b) Free papain induces activation in the production of nitric oxide (Fig 7a), indicating that it is phagocytosed and processed However, immobilized papain does not act in the process of induction of nitric oxide The non-induction of nitric oxide by immobilized papain suggests that this nanoparticle model does not induce macrophage activation This can be positive when it is desired to construct a system that is not enzyme could not be estimated because the values of cellular viability were greater than 80 % The results of the assay with the immobilized enzyme are shown in Fig 5d The nanomaterial demonstrated cytotoxicity at the last four concentrations, and this behavior confirmed that the CC50 was 488.3 μg.mL−1 Pati, Das, Mehta, Sahu, and Sonawane (2016) showed that the cellular viability of macrophages exposed to ZnO NPs is dependent on concentration and that it decreases by 80 % at a concentration of 100 μg.mL−1 of ZnO NPs (Pati et al., 2016) This is because ZnO NPs cause apoptosis (Zhang et al., 2012) and cellular autophagy (Zhang et al., 2012), contributing to the elevated cytotoxicity of immobilized papain in the presence of ZnO NPs Macrophages are essential elements of host cell defense systems and participate in the purification process of any foreign element, degrading Carbohydrate Polymers 243 (2020) 116498 A.M.B.F Soares, et al purified by the phagocytic system As a way of confirming that macrophages are activated by papain immobilized, lysosomal activity, and phagocytic capacity assays were performed (Fig 7c-f) It has been found that immobilized papain does not induce lysosomal activity and increases phagocytic capacity These results are important because they suggest that the phagocytic system does not have a great ability to purify this model of nanoparticles, demonstrating that once injected, it may have a longer duration 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(2013) Preparation of welldispersed gold/magnetite nanoparticles embedded on cellulose nanocrystals for efficient immobilization of papain enzyme ACS Applied Materials & Interfaces, 5(11), Conclusion The immobilization of papain enzyme on a hybrid support containing ZnO/chitosan was successfully performed, yielding nanotriangular structures with a size of 150 nm, as revealed by TEM Collagen formation and casein degradation tests indicated that the immobilized enzyme system had not lost its proteolytic capacity, favoring the maintenance of enzymatic activity Bionanomaterial (with papain) not activate the cell phagocytic system, which is promising for biomedical applications that use the debridement and healing properties of papain, chitosan scarring, and the bacteriostatic actions of zinc oxide, owing to the low cytotoxicity demonstrated by the in vitro evaluation of cytotoxicity The proposed system is a low-cost alternative that can also be applied to the immobilization of other enzymes Author contributions All authors conceived and designed the experiments CRediT authorship contribution statement Aurileide M.B.F Soares: Conceptualization, Methodology, Data curation, Writing - original draft Lizia M.O Gonỗalves: Conceptualization, Methodology Ruanna D.S Ferreira: Conceptualization, Methodology Jeerson M de Souza: Resources Raul Fangueiro: Resources Michel M.M Alves: Methodology Fernando A.A Carvalho: Methodology Anderson N Mendes: Conceptualization, Methodology, Data curation, Writing - original draft, Supervision, Project administration, Funding acquisition Welter Cantanhêde: Conceptualization, Methodology, Data curation, Writing original draft, Supervision, Project administration, Funding acquisition Declaration of Competing Interest The authors declare no conflict of interest Acknowledgments The financial support from CNPq (310678/2014-5), FAPEPI and CAPES (Rede nBioNet) is gratefully acknowledged Appendix A Supplementary data Supplementary material related 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denaturation and aggregation during cold storage Spectrochimica Acta Part A, Molecular and Biomolecular Spectroscopy, 150, 238–246 https://doi.org/10.1016/j.saa.2015.05 061 Regiel-Futyra, A., Kus-Liśkiewicz, M., Wojtyła, S., Stochel, G., & Macyk, W (2015) The 10 ... nitric oxide) : (a) free papain and (b) Immobilized papain; evaluation of lysossomal volume: (c) free papain and (d) Immobilized papain; evaluation of phagocytic capacity: (e) free papain and (f)... Kumar, A B., Varadaraj, M C., Lalitha, R G., & Tharanathan, R N (2004) Low molecular weight chitosans: Preparation with the aid of papain and characterization Biochimica et Biophysica Acta, 1670(2),... as a percentage (Tumer et al., 2007) For lysosomal activity, peritoneal macrophages were plated and incubated with pure papain and immobilized papain (at a concentration Results and discussion