Ferroelectrics

ISSN: 0015-0193 (Print) 1563-5112 (Online) Journal homepage: https://www.tandfonline.com/loi/gfer20

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 ARTICLE HISTORY

Bacterial infection is a major challenge for contemporary medicine, Received 24 July 2019
and antimicrobial hydrogels have been extensively studied for their Accepted 30 January 2020
excellent antimicrobial activity. However, the antibacterial agent also
KEYWORDS
has high cytotoxicity and hemolytic activity while being significantly
ZIF-90; antibacterial;
antibacterial. In this paper, we prepared a hydrogel by simple heat- hydrogel; biocompatible
ing-cooling of ZIF-90 doped agar solution, which has obvious anti-
bacterial 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 patho-
genic 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 nor-
mal cells while killing microorganisms, and even adversely affect their biological func-
tions [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 proper-
ties and best antibacterial properties in various metal ions [3], but it may have a detri-
mental effect on cellular responses and maybe potentially toxic even at high
concentrations [4]. However, besides the excellent antibacterial property, Zn promotes

CONTACT Bing Yu yubingqdu@yahoo.com

ß 2020 Taylor & Francis Group, LLC
 FERROELECTRICS 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 hydro-
gel 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(CH3COOH)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. Gram-
positive 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 bacter-
ial 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 dis-
solved 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
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 inhib-
ition zone was observed after incubation at 37 C for 12 hours.
14/[166] Y.-C. TIAN ET AL.

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 intra-
cellular 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 super-
natant 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% penicillin-
streptomycin 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 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 absorb-
ance relative to that of the control.
 FERROELECTRICS 15/[167]

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 emer-
gence 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 success-
fully coated on the agar gel without any chemical reaction. It can be seen from Figure
2b, the 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 dis-
played in the SEM image and appears smoother than the ZIF-90 hydrogels(Figure 3c),
16/[168] Y.-C. TIAN ET AL.

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).

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
 FERROELECTRICS 17/[169]

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 indi-
cated 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 incuba-
tion with ZIF-90 hydrogels for 12 h are shown in Figure 5. Calcein-AM, which fluores-
ces 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 bac-
teria fluoresced red, revealing the excellent antimicrobial activities of the ZIF-
90 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-2-
thiazolyl)-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 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 biomate-
rials. As shown in Figure 6b. the hemolysis ratio with hydrogels concentrations ranging
18/[170] Y.-C. TIAN ET AL.

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.

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.
 FERROELECTRICS 19/[171]

from 300 to 5000 mg mL-1 was lower than 35%, indicating the excellent blood compati-
bility 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 bac-
teria (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 bacter-
ial 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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