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

Carbohydrate Polymers 243 (2020) 116498

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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, Jeerson 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.

1. 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: (W. Cantanhêde).
/>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 7 was reached and was then oven-dried for 2
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
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−1 papain solution were
added and kept under agitation for 2 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 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−1 in
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−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 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 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 5 min 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.

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 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 5 × 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 min at 25 °C. After decantation, the precipitate was washed three
times with ultrapure water, oven-dried at 100 °C for 2 h, and finally
calcined in a muffle furnace for 2 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, 3 mL of water were added to the
negative control; for tube B, 3 mL of free papain were added at 1
mg.mL−1 for the positive control, and 3 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 4 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 min
under constant magnetic stirring to disperse the solution. Subsequently,
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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 min. 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
min 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 min 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 6 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 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 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 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, 100 μL 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% CO2 for 4 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 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 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

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 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 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 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
3


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Fig. 1. 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 1 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.
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Fig. 3. 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. 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.
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. 3 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,
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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.

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),
6


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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. 5 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. 6 shows the activity of free and immobilized papain in the cell

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.

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. 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.
7


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A.M.B.F. Soares, et al.

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.

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
8

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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 in the circulatory system or any cell tissue.

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4. 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) 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 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 to this article can be found, in the
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