Ferroelectrics

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Agar-based ZIF-90 antibacterial

hydrogels for biomedical applications

Yong-Chang Tian, Cai-Cai Jiao, Song Wang, Hai-Lin Cong,

You-Qing Shen & Bing Yu

To cite this article: Yong-Chang Tian, Cai-Cai Jiao, Song Wang, Hai-Lin Cong, You-
Qing
Shen & Bing Yu (2020) Agar-based ZIF-90 antibacterial hydrogels for biomedical applications,
Ferroelectrics, 563:1, 12-20, DOI: 10.1080/00150193.2020.1760605
To link to this article:
https://doi.org/10.1080/00150193.2020.1760605

Published online: 23 Jul

2020.

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FERROELECTRICS
2020, VOL. 563, 12–20
https://doi.org/10.1080/00150193.2020.1760605

Agar-based ZIF-90 antibacterial hydrogels for biomedical

applications
Yong-Chang Tiana, Cai-Cai Jiaoa, Song Wanga, Hai-Lin Conga,b, You-Qing Shena, and Bing
Yua,b
a
Institute of Biomedical Materials and Engineering, College of Chemistry and Chemical Engineering,
Affiliated Hospital of Qingdao University, College of Materials Science and Engineering, Qingdao
University, Qingdao, China; bState Key Laboratory of Bio-Fibers and Eco-Textiles, Qingdao University,
Qingdao, China

ABSTRACT
Bacterial infection is a major challenge for contemporary medicine, and ARTICLE HISTORY
antimicrobial hydrogels have been extensively studied for their Received 24 July 2019
excellent antimicrobial activity. However, the antibacterial agent also Accepted 30 January 2020
has high cytotoxicity and hemolytic activity while being significantly KEYWORDS
antibacterial. In this paper, we prepared a hydrogel by simple heating- ZIF-90; antibacterial; hydrogel;
cooling of ZIF-90 doped agar solution, which has obvious antibacterial biocompatible
activity against Gram-positive and Gram-negative bacteria. The test
results show that the hydrogel has a uniform and stable structure and
does not exhibit cytotoxicity and hemolytic activity while maintaining
good bacteriostatic properties, so as to achieve the function of bacterial
infection treatment and would healthcare.

1. Introduction
The bacterial infection is a local or systemic inflammatory reaction caused by pathogenic
bacteria or opportunistic pathogens, which is a serious problem in the field of biomedicine.
As a highly effective and low toxic treatment, antibiotics have been applied to various
bacterial infections since they were first discovered in 1928 [1]. However, a serious
problem remains that the use of antibiotics has led to the emergence of multidrug-resistant
microorganisms that are difficult to fight [2]. Challenged by the ever-growing threats from
drug-resistant pathogenic microorganisms, there has been increasing attention on
developing antibacterial materials to defend against infections. Unfortunately, antibacterial
ingredients often have adverse effects on surrounding normal cells while killing
microorganisms, and even adversely affect their biological functions [1]. Therefore,
designing materials that meet antimicrobial and biocompatibility requirements have always
been a challenging task.
For example, Agþ was widely recognized for its broad-spectrum antibacterial properties
and best antibacterial properties in various metal ions [3], but it may have a detrimental
effect on cellular responses and maybe potentially toxic even at high concentrations [4].
However, besides the excellent antibacterial property, Zn promotes
 Y.-C. TIAN ET AL.
CONTACT

yubingqdu@yahoo.com
Bing Yu
2020 Taylor & Francis Group, LLC
13/[165]

protein synthesis and adsorption and provides a positive effect on cell adhesion and
proliferation depending on its content [5]. A hydrogel is a three-dimensional porous
material composed of physically or chemically cross-linked polymer chains [6]. Hydrogels
have been extensively studied as antibacterial material, and after carefully selecting the
monomers and cross linkers, the hydrophilicity and porosity of the hydrogel can be used
for antimicrobial applications [7]. In addition, certain types of hydrogels also have inherent
antimicrobial properties [8].
In the present work, the ZIF-90 hydrogel was prepared by using agar as the Hydrogel
matrix, with the additional incorporation of ZIF-90 to enhance its antimicrobial activity.
Staphylococcus aureus (S. aureus), a common pathogen in the suppurative inflammation
and open wound infection, and Escherichia coli (E.coli) were selected to systematically test
the antibacterial efficacy of the prepared materials. L929 cells were selected to test the
cytotoxicity of the prepared ZIF-90 hydrogels. Hemolytic of the hydrogels is also detected
as a biocompatibility evaluation parameter.

2. Experiments
2.1. Preparation of ZIF-90
For the preparation of nano ZIF-90, 20 mL imidazolate-2-carboxaldehyde (2-ICA) (0.4 M)
in DMSO was added into 20 mL Zn(CH 3COOH)22H2O (0.4 M) in DMF with stirring for
10 min. Then, another 40 mL DMF was added for another 10 min stirring. The mixture
was then washed by ethanol for three times and dried in vacuum for 24 h [9].

2.2. Preparation of ZIF-90 composite hydrogels

For the preparation of ZIF-90 hydrogels, 0.03 g ZIF-90 powder and 0.2 g of agar powder
were uniformly dispersed in 10 ml deionized water. ZIF-90 hydrogels were obtained after
the mixture was heated to 60 C and naturally cooled.

2.3. Antibacterial assay

Antimicrobial properties of ZIF-90 were detected by the cylinder plate method.
Grampositive bacteria, S. aureus (ATCC 29213), and Gram-negative bacteria, E. coli
(ATCC 8739) were selected for the experiment, and the experiments were performed
according to the methods reported elsewhere [10]. E.coli and S. aureus were cultured on
LB agar plates at 37 C for 24 h, A single bacteria colony was picked and inoculated into
LB broth, then incubated at 37 C with shaking at 200 rpm overnight. Next, 200ul of
bacterial suspension was transferred to 35 ml of fresh LB broth and incubated at 37 C with
shaking at 300 rpm until reaching logarithmic phase.(i) 10 ml sterile distilled water
dissolved with 0.25 g LB broth and 0.13 g Aa was evenly spread in the culture dish as the
bottom layer and (ii) 5 ml sterile distilled water dissolved with 0.2 g LB broth, 0.06 g Aa
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and 200ul bacterial suspension was spread as the top layer. Hydrogel with a circular
diameter of 10 mm was placed on the culture medium, and the bacterial growth inhibition
zone was observed after incubation at 37 C for 12 hours.

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2.4. Live/dead bacterial assay

The Calcein-AM/PI Double Stain Kit was used in the experiment. Calcein-AM is a dye that
stains living bacteria [11]. When it enters the cell, calcein-AM is cleaved by intracellular
esterases to produce calcein, which fluoresces in green (Ex ¼ 490 nm, Em ¼ 515 nm). In
contrast, PI dye stains only dead bacteria with damaged membranes because it is not
celling permeable. It fluoresces in red when it interacts with the nucleus (Ex ¼ 535 nm, Em
¼ 617 nm). The bacteria/fungi were allowed to grow in the broth for 20 h to reach the mid-
log phase and then incubated with hydrogels(100 mg/mL) at 37 C with shaking at 150 rpm
overnight. After centrifuging at 3000 rpm for 5 min. the supernatant was removed, and the
bacterial pellets were washed three times with buffer. Calcein-AM/PI (100 lL) was added
to the bacterial suspensions (200 lL) and incubated in the dark at room temperature for 25
min. The samples were imaged with a laser scanning confocal microscopy.

2.5. Hemolysis test

A hemolytic activity assay was conducted to evaluate the blood compatibility of the
hydrogels. The hydrogels were incubated with red blood cells (RBCs) from rats, and its
HC50 was calculated. HC50 is defined as the concentration that causes 50% hemolysis of
RBCs. The test was performed according to an existing reference [12]. First, hydrogels at
concentrations from 125 to 2000 lg/mL were placed in a 24-well plate. Then, 1 mL of
erythrocyte suspension (4% v/v in PBS) was added to each well, with PBS as negative
control and 1%Triton x-100 as a positive control. After incubation for 1 h at 37 C, the plate
was centrifuged at 3000 rpm for 5 min, and 50 lL of supernatant was transferred to a 96-
well plate. The absorbance was measured at 540 nm. The hemolytic activity was calculated
as follows:

Hemolysis ð%Þ ¼ ðA–A0Þ=ðA100–A0Þ 100%

in which A, A0, and A100 represent the absorbance of hydrogels and the negative and
positive controls, respectively.

2.6. Cytocompatibility assay

L929 cells were cultured in RPMI-1640 supplemented with 10% FBS and 1%
penicillinstreptomycin in a humidified5% CO2 atmosphere [13]. After ultraviolet
sterilization, the preprepared hydrogels was immersed in RPMI-1640 for 24 h. The cells
were seeded into a 96-well tissue culture plate at a density of 6 103 cells/well and
incubated in 100 lL of RPMI-1640/well for 24 h. Then, the cell culture medium was
replaced with 200 lL serial dilutions of hydrogel soaking solution in a fresh growth
medium. After 48 h, 20 lL of MTT was added to each well of the plate, and the plate was
 Y.-C. TIAN ET AL.
incubated in a cell culture incubator at 37 C for 4 h. After removing the culture media,
formazan crystals were completely dissolved in 200 lL DMSO, and the absorbance values
were measured in triplicate at a test wavelength of 492 nm. The cell viability was
expressed as absorbance relative to that of the control.

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Figure 1. a) Scanning electron microscopy (SEM) diagram of ZIF-90. b) Energy dispersive spectrum
(EDS) of ZIF-90.

3. Result and discussion

3.1. Preparation of ZIF-90
The ZIF-90 particles within the 80 to 200 nm range were successfully synthesized via a
room-temperature method [14], as shown in Figure 1a. Scanning electron microscopy
(SEM) revealed that isolated NPs have an average diameter of 80–300 nm. The emergence
of the peak of zinc from the EDS spectrum confirmed that the zinc content in ZIF reached
16.3% (Figure 1b).

3.2. Preparation of ZIF-90 composite hydrogels

As shown in Figure 2a, the agar constitutes a hydrogel block frame and the hydrogels were
obtained after cooling the heated agar solution, and ZIF-90 particles are successfully
coated on the agar gel without any chemical reaction. It can be seen from Figure 2b, the
 FERROELECTRICS
prepared hydrogel formed at the bottom of the volumetric flask and does not deform with
the turning, which proves that the gel has good mechanical strength. A hydrogel is a three-
dimensional network structure formed by crosslinking a hydrophilic polymer, as shown in
Figure 3a,b The lamellar structure of the agar gel is clearly displayed in the SEM image
and appears smoother than the ZIF-90 hydrogels(Figure 3c),

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Figure 2. a) Schematic diagram of ZIF-90 hydrogel.b) Optical images of the prepared hydrogels.

Figure 3. Scanning electron microscopy (SEM) diagram of a,b) surface of the agar hydrogel. c)
surface of the ZIF-90 hydrogel. d) ZIF-90 particles on the surface of the hydrogel.

and the ZIF-90 partials absorbed can be clearly observed on the surface of the hydrogel
(Figure 3d).
 Y.-C. TIAN ET AL.
3.3. Antibacterial assay
The antibacterial activity of the hydrogels was determined by a Plate diffusion method
against both E.coli and S.aureus. The hydrogel sample with a diameter circular of 1 cm

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Figure 4. Antibacterial property test with selected bacterial a) E.coli, b) S.aureus, of the hydrogels
by plate diffusion method. The inhibition zone of the hydrogel against E.coli2.5 mm, and
S.aureus5.5 mm.

was placed directly onto the surface of the bacterial media of E.coli and S.sureus
respectively. After incubated at 37 C for 12 h, the emerge of the inhibition zone indicated
that hydrogel showed excellent activity against both E.coli and S.aureus (Figure 4a and b).
Moreover, as we can see from Figure 4, the inhibition zone of the gel against S.aureus was
5.5 mm, much larger than which against E.coli (2.5 mm). The possible speculation of this
case is that compared with E.coli (Gram-negative genus) , there are more viscous proteins,
and the peptidoglycan content in the cell wall of the S.aureus (Gram-positive genus), which
makes it easier to adsorb zinc ions.
The live/dead microbial viability assays of E.coli and S.aureus before and after
incubation with ZIF-90 hydrogels for 12 h are shown in Figure 5. Calcein-AM, which
fluoresces upon reaction with intracellular esterase, stains live cells (green), while
propidium iodide (PI), which binds to the DNA of dead membrane-compromised cells,
stains dead cells (red). Almost all intact bacteria fluoresced green, whereas hydrogels-
treated bacteria fluoresced red, revealing the excellent antimicrobial activities of the ZIF90
hydrogels.

3.4. Cytocompatibility assay

The ideal biomedical material should first be nontoxic or minimally toxic to the body. To
assess the cytocompatibility of the ZIF-90 hydrogels, a standard 3-(4,5-dimethyl-
2thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide (MTT) assay with L929 cells was
employed. The L929 cells were incubated with different concentrations of hydrogels for 24
 FERROELECTRICS
h (Figure 6a). Encouragingly, the cell viability values with hydrogels concentrations
ranging from 625 to 10000 mg mL-1 were higher than 80%, demonstrating the excellent
biocompatibility of the ZIF-90 hydrogels.
Hemolysis is another important index for evaluating the biocompatibility of
biomaterials. As shown in Figure 6b. the hemolysis ratio with hydrogels concentrations
ranging

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Figure 5. Live/dead microbial viability assay of E.coli (a series), and S.aureus (b series) before (a1-
a2) and after (b1-b2) incubation with ZIF-90 hydrogels for 12 h.
 Y.-C. TIAN ET AL.

Figure 6. Biocompatibility of ZIF-90 hydrogels. a) Cell viability of L929 cells as a function of

hydrogels after incubation for 24 h. b) Hemolytic activity of hydrogels as a function of
concentration. Positive control: T-100.
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from 300 to 5000 mg mL-1 was lower than 35%, indicating the excellent blood
compatibility of the ZIF-90 hydrogels.

4. Conclusion
In summary, hydrogels with excellent antibacterial property and biocompatibility were
prepared via simply heated and cooled with the ZIF-90-agar solution. The hydrogels were
found to inactive both Gram-positive bacteria (S.aureus) and gram-negative bacteria
(E.coli). Importantly, they were shown to be nontoxic toward human erythrocytes and
caused no organ toxicity when applied internally. The biocompatible hydrogels developed
herein thus can be used as a safe and effective material to prevent bacterial infection.

Funding
This study was funded by the National Natural Science Foundation of China (21675091, 21874078),
the Taishan Young Scholar Program of Shandong Province (tsqn20161027), the Major Science and
Technology Innovation Project of Shandong Province (2018CXGC1407), the
Key Research and Development Project of Shandong Province (2016GGX102028,
2016GGX102039, 2017GGX20111), the Innovation Leader Project of Qingdao (168325zhc), and
the First Class Discipline Project of Shandong Province.

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