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.

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Carbohydrate Polymers journal homepage:www.elsevier.com/locate/carbpol

Immobilization of papain enzyme on a hybrid support containing zinc oxide

Aurileide M.B.F Soaresa, Lizia M.O Gonçalvesa, Ruanna D.S Ferreirab, Je fferson 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

b 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

A R T I C L E I N F O

Keywords:

Enzyme immobilization

Papain

Zinc oxide

Chitosan

Hybrid materials

Clinical application

A B S T R A C T

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 im-mobilized 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 bio-medical applications in diseases such as psoriasis and wounds

1 Introduction

Chitosan is a natural polysaccharide composed of D-glucosamine

linked toβ- (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

poly-saccharide is suitable for clinical applications because of its low

toxi-city, 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

na-nostructures, 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 re-sidues, 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 asso-ciated 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 deb-ridement 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

https://doi.org/10.1016/j.carbpol.2020.116498

Received 23 February 2020; Received in revised form 20 April 2020; Accepted 20 May 2020

☆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)

Available online 26 May 2020

0144-8617/ © 2020 Published by Elsevier Ltd

T

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-che-mical characteristics such as chephysical-che-mical and mechanical resistance,

pro-cessability, 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

ex-pensive and time consuming Therefore, studies for new hybrid

nano-materials 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

pa-pain 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 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 PolymarCiência e

Nutrição S/A, located in the Technological Development Park at

Federal University of Ceará (Padetec-UFC Brazil), was used in the

preparation of 5 × 10−3g.L-1of 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

de-scribed 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 reactionflask containing 25 mL of zinc

acetate dehydrate 0.1 mol.L-1to form a colloidal suspension with a

white color The mixture was kept under constant magnetic stirring for

90 min at 25 °C After decantation, the precipitate was washed three

times with ultrapure water, oven-dried at 100 °C for 2 h, andfinally

calcined in a muffle furnace for 2 h at 300 °C at a heating rate of 10

°C.min−1

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 reactionflask Then, 350 mg of ZnO NPs prepared from the

pre-vious step were added to the chitosan solution and held for 30 min

under constant magnetic stirring to disperse the solution Subsequently,

25 mL of 1.0 mol.L−1NaOH were added to the mixture to precipitate the chitosan on the ZnO NPs The white precipitate was washed with ultrapure water until a pH 7 was reached and was then oven-dried for 2

h at 100 °C and reserved for immobilization

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 col-laborators, 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

2 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 6 mg.mL−1papain solution were added and kept under agitation for 2 h at room temperature for im-mobilization to occur by chemical binding The slightly yellowish precipitate was washed three times with ultrapure water and finally oven-dried at 40 °C for 1 h

2.5 Characterizations

The formation of the ZnO/chitosan support was investigated by reaction with ninhydrin For this purpose, a dispersion of 5 mg.mL−1in phosphate-buffered saline (PBS) (pH 7.0) was prepared, followed by 1

mL of a solution of 3% (m/v) ninhydrin in ethanol The mixture was heated for 1 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−1over 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 1 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 tempera-ture, 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 measure-ments, the dispersions of the nanomaterials were prepared at a con-centration of 0.04 mg.mL−1using the KCl solution (Dinâmica, Brazil) of 1.0 × 10-3mol.L−1and left to stir in an ultrasonic bath for 5 min at 25

°C Solutions of HCl (Dinâmica, Brazil) and KOH (Dinâmica, Brazil) with a concentration of 0.1 mol.L−1each were used to adjust the pH of each dispersion The zeta potential was registered as the mean value of three measurements

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, 3 mL of water were added to the negative control; for tube B, 3 mL of free papain were added at 1 mg.mL−1for the positive control, and 3 mL of immobilized papain were added at 10 mg.mL−1in tube C All tubes remained under constant stirring until homogenization occurred, and then were cooled for 4 h The proteolysis capacity of the immobilized enzyme was evaluated by observing gel formation

2.6.2 Degradation of casein by immobilized papain

To evaluate the degradability of casein after the immobilization of

papain, a 6 mg.mL−1 dispersion of immobilized papain system was

initially prepared and transferred to a reactionflask 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 6 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

mac-rophages were isolated from BALB/c mice (males or females,

5–6-week-old (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

2.8 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

con-centrations of 12.5, 25, 50, 100, 200, 400, and 800 μg.mL−1

Ery-throcyte arterial blood (Ovis aries) was incubated for 1 h at 37 °C with

different samples The samples were centrifuged at 1610 × g for 5 min

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, 100μL of Roswell Park Memorial Institute (RPMI)

1640 culture medium (Thermo Fisher Scientific, Waltham, MA, USA)

supplemented with 2 × 105 macrophages per well were added and

incubated at 37 °C with 5% CO2for 4 h to allow for cell adhesion Two

washes with RPMI 1640 were performed to remove the cells that did

not adhere Then, 100μL of RPMI medium with the free or immobilized

enzyme diluted to the concentrations of 800 to 6.25 μg.mL−1were

added, in triplicates The wells were incubated for 48 h Then, 10μL 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

in-cubated at 37 °C and 5% CO2 After discarding the medium, 100μL of

dimethyl sulfoxide (DMSO) were added to all wells The plate was

shaken for 30 min 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 2 ×

105 macrophages per well, with solutions of pure papain and

im-mobilized papain at concentrations of 800 to 6.25μg.mL−1, in

tripli-cates 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

con-centrations of 1, 5, 10, 25, 50, 75, 100, and 150μM diluted in RPMI

1640 medium For the nitrite dosage, equal parts of the prepared

so-lutions (50μL 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 %

naphthyle-nediamine 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

of 80μg.mL−1) After 48 h of incubation at 37 °C and 5% CO2, 10μL of 2% neutral red DMSO solution were added, followed by incubation for

30 min Then, the supernatant was discarded, and the wells were wa-shed with 0.9 % saline at 37 °C, and 100μL of extraction solution were added to solubilize the neutral red present inside the lysosomal secre-tory vesicles The evaluation took place in a spectrophotometer after 30 min at 550 nm

For the evaluation of the phagocytic capacity, peritoneal macro-phages were plated and incubated with the test solution After 48 h incubation at 37 °C and 5% CO2, 10μL of stained zymosan solution were added and incubated for 30 min at 37 °C followed by the addition

of 100μL 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, 100μL of extraction solution were added and the solution was analyzed using a spectrophotometer at 550 nm

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 sig-nificance

3 Results and discussion

3.1 Synthesis, molecular structure, and crystallinity

Fig 1 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 chit-osan was dissolved in acetic acid (pH 5.0), resulting in the protonation

of -NH2groups 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 be-cause of the interaction between the oxygen atoms of the NPs with the nitrogen and oxygen atoms of chitosan The -NH2groups 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 mate-rials 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

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 7 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 crystal-lographic 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

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

frac-tion For this calculation, we considered the ranges of 10° to 30° and 30°

to 90° as the amorphous and the crystalline fraction, respectively

Table 1 shows the crystallinity indices for ZnO NPs, ZnO/chitosan

support substrate, and the enzyme immobilized on the ZnO/chitosan

support

=

+

Ic Ia

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

crys-tallinity 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

ap-proximately 23° confers a higher degree of crystallinity to the

im-mobilized 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

3.2 Spectroscopic investigation, reactivity, and supramolecular arrangement

Molecular absorption spectroscopy analysis in the visible, ultra-violet (UV) region (UV–vis) was performed to investigate the formation

of the materials in colloidal suspensions The formation of nanoparti-culate 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

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 orbitalσ (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 -NH2groups present in the chitosan structure Absorption at 362 nm was also observed, which was attrib-uted 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 scat-tering, 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

Fig 1 Schematic representation for the fabrication of immobilized papain system

Under certain conditions, ninhydrin reacts with free -NH2groups to

form a purple-colored compound,

diketohydrindylidene-diketohy-drindamine, known as Purple of Ruhemann This reaction can be

con-firmed by UV–vis, since Ruhemann’s purple exhibits a characteristic

absorption at around 568 nm, as shown inFig 2b (Lu, 2013) The inset

inFig 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 3shows the FTIR spectra of pure

chit-osan, free papain, ZnO NPs, ZnO/chitchit-osan, and immobilized papain For

chitosan, the peak at around 3350 cm−1was 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 ofυC-H, while the strain vibrations at 1658,

1583, 1418, and 1315 cm−1were related to amide I,™N-H deformation present in the -NH2group, axial deformationυC-N, and amide III, re-spectively

The vibrations at 1074 and 1034 cm−1were 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-1and the peak at 2931 cm−1were attrib-uted to the OH,υN-H of the secondary amine, and υassC-H (sp3), re-spectively (Fig 3b) The FTIR spectrum of papain also exhibited stret-ches 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−1from the C–S stretches of sulfide and disulfide [132] For ZnO NPs, vibration at 3646 cm−1was 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 theυZn-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 de-formation at 497 cm−1that 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 im-mobilization of papain in the proposed substrate was confirmed by FTIR.Fig 3e shows the increase in absorption at 1660 cm−1for 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 -NH2groups on the support and the papain, since the nucleo-philic amine groups attack the aldehyde groups present in the glutar-aldehyde 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 2 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 1

Index of crystallinity of the synthesized materials

Fig 3 FTIR spectra for (a) Chitosan, (b) Free papain, (c) ZnO NPs, (d) ZnO/ Chitosan, (e) ZnO/Chitosan-glutaraldehyde and (f) immobilized papain

aggregates (Salavati-Niasari, Mir, & Davar, 2009) The particle size

distribution histogram of the nanotriangles (n = 113 particles)

re-vealed 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 byKumar et al (2019)andRodrigues 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

esti-mate 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),

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 ZnO2−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 demon-strates 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

Fig 4 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

papain on the surface, increasing its hydrodynamic diameter In

addi-tion, 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 Thesefindings are in agreement with the TEM images results,

in which the decrease in ZnO nanoparticle size after chitosan and

en-zyme 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

en-zyme 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

pro-cess

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 5shows 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

accom-panied by spectrophotometric results in the UV–vis region It is

inter-esting 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 6shows the activity of free and immobilized papain in the cell

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 CC50for the

Fig 5 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

Fig 6 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

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 inFig 5d

The nanomaterial demonstrated cytotoxicity at the last four

con-centrations, 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

100μg.mL−1of 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

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 pro-duced during the process of phagolysosome formation (Cape & Hurst,

2009;Tumer et al., 2007)

Thus, nitrite production activity was evaluated to verify the beha-vior 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

Fig 7 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

purified by the phagocytic system As a way of confirming that

mac-rophages 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 in the circulatory system or any cell tissue

4 Conclusion

The immobilization of papain enzyme on a hybrid support

con-taining ZnO/chitosan was successfully performed, yielding

nano-triangular 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,

fa-voring the maintenance of enzymatic activity Bionanomaterial (with

papain) do 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

alter-native 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 Gonçalves:

Conceptualization, Methodology Ruanna D.S Ferreira:

Conceptualization, Methodology Jefferson 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 to this article can be found, in the

online version, at doi:https://doi.org/10.1016/j.carbpol.2020.116498

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