gels

Article
Synthesis and Characterization of Hydrogel Droplets
Containing Magnetic Nano Particles, in a Microfluidic
Flow-Focusing Chip
Fereshteh Moharramzadeh 1 , Seyyed Ali Seyyed Ebrahimi 1, *, Vahid Zarghami 2 , Zahra Lalegani 1
and Bejan Hamawandi 3, *

1 Advanced Magnetic Materials Research Center, School of Metallurgy and Materials, University of Tehran,
Tehran 11155 4563, Iran; [email protected] (Z.L.)
2 Department of Materials and Metallurgy, Faculty of Mechanical and Energy Engineering,
Shahid Beheshti University, Tehran 16589 53571, Iran; [email protected]
3 Department of Applied Physics, KTH Royal Institute of Technology, SE-106 91 Stockholm, Sweden
* Correspondence: [email protected] (S.A.S.E.); [email protected] (B.H.)

Abstract: Magnetic hybrid hydrogels have exhibited remarkable efficacy in various areas, particu-
larly in the biomedical sciences, where these inventive substances exhibit intriguing prospects for
controlled drug delivery, tissue engineering, magnetic separation, MRI contrast agents, hyperthermia,
and thermal ablation. Additionally, droplet-based microfluidic technology enables the fabrication
of microgels possessing monodisperse characteristics and controlled morphological shapes. Here,
alginate microgels containing citrated magnetic nanoparticles (MNPs) were produced by a microfluidic
flow-focusing system. Superparamagnetic magnetite nanoparticles with an average size of 29.1 ± 2.5 nm
and saturation magnetization of 66.92 emu/g were synthesized via the co-precipitation method. The
hydrodynamic size of MNPs was changed from 142 nm to 826.7 nm after the citrate group’s attachment
led to an increase in dispersion and the stability of the aqueous phase. A microfluidic flow-focusing chip
was designed, and the mold was 3D printed by stereo lithographic technology. Depending on inlet fluid
Citation: Moharramzadeh, F.; Seyyed rates, monodisperse and polydisperse microgels in the range of 20–120 µm were produced. Different
Ebrahimi, S.A.; Zarghami, V.; conditions of droplet generation in the microfluidic device (break-up) were discussed considering the
Lalegani, Z.; Hamawandi, B. model of rate-of-flow-controlled-breakup (squeezing). Practically, this study indicates guidelines for
Synthesis and Characterization of
generating droplets with a predetermined size and polydispersity from liquids with well-defined
Hydrogel Droplets Containing
macroscopic properties, utilizing a microfluidic flow-focusing device (MFFD). Fourier transform
Magnetic Nano Particles, in a
infrared spectrometer (FT-IR) results indicated a chemical attachment of citrate groups on MNPs and
Microfluidic Flow-Focusing Chip.
the existence of MNPs in the hydrogels. Magnetic hydrogel proliferation assay after 72 h showed a
Gels 2023, 9, 501. https://doi.org/
10.3390/gels9060501
better rate of cell growth in comparison to the control group (p = 0.042).

Academic Editor: Bjørn Torger Keywords: microfluidic systems; droplet; flow-focusing system; alginate; magnetic hydrogel; magnetite
Stokke

Received: 31 March 2023

Revised: 11 June 2023
Accepted: 16 June 2023 1. Introduction
Published: 19 June 2023 Hydrogels are commonly used in biomedical engineering due to their hydrated nature
and adjustable properties (biocompatibility, chemical, and mechanical characteristics) close
to the natural extracellular matrix (ECM) [1]. Nonetheless, there are still problems when
it comes to the use of hydrogels in fields such as 3D tissue construction engineering and
Copyright: © 2023 by the authors.
active targeting in drug delivery given the lack of controllability, actuation, and rapid
Licensee MDPI, Basel, Switzerland.
This article is an open access article
response properties [2].
distributed under the terms and
Recently, a wide range of applications and active properties of magnetic hydrogels
conditions of the Creative Commons have introduced them as a novel biocomposite. These kinds of hydrogels are utilized
Attribution (CC BY) license (https:// in tissue engineering [3], image enhancement [4], sorting and separation [5], immunoas-
creativecommons.org/licenses/by/ says [6], drug delivery and release [7], immobilization of enzymes [8], cancer treatment by
4.0/). chemotherapy and hyperthermia [9], and soft actuation [10].

Gels 2023, 9, 501. https://doi.org/10.3390/gels9060501 https://www.mdpi.com/journal/gels

Gels 2023, 9, 501 2 of 14

Knowing that cells exposed to magnetic fields or materials with magnetic properties
can regulate several biological responses, Filippi et al. aimed to create an advanced bone
regeneration platform using magnetic actuation to prime stromal vascular fraction (SVF)
cells. Magnetized hydrogels, created from the co-assembly of cells, polyethylene glycol
(PEG), and PEG-coated superparamagnetic iron oxide NPs, were used to enhance SVF
biological function under an external magnetic field [11].
Furthermore, anisotropic tissue patterns in bioengineered constructs are challeng-
ing. Using responsive magnetic hydrogels for 3D bioprinting of scaffolds and magneto-
mechanical actuation can create an ideal strategy for anisotropic mechanosensitive tissue
engineering [12].
Magnetic targeting enables the targeting of a tumor and hence prevents the systematic
distribution of cytotoxic compounds; this leads to an enhancement of drug uptake at the
target site inside the body and lowers the required doses for effective treatment. Further-
more, it was shown that magnetic hydrogels along with the alternating magnetic field
could be used as hyperthermia treatment by inducing tumor regression [13,14].
In practice, micron-sized and morphologically homogeneous monodisperse composite
particles are preferred since they have reproducible and consistent behavior. Particles are
required to be biocompatible and biodegradable to be used in biomedical applications [15].
Recently, some microfluidic devices were utilized for the fabrication of polymeric
microparticles that are widely utilized in the delivery of cells and drugs, 3D-cell culture,
tissue engineering, and immunoassays [13,14,16,17].
The control of the size, shape, and polydispersity along with the precise generation and
repeatability of droplet operations, have made a droplet-based microfluidic system a potent
high throughput platform for biomedical research and applications. In addition to being
used as micro reactors, droplet-based systems have also been used to directly synthesize
particles and encapsulate many biological entities for biomedicine and biotechnology
applications [18,19].
The MFFD, T-junctions, and those that liquid threads break on the terraces of the
microchannel which the dispersed phase, break upon their intersection with the continuous
phase at the most effective area on-chip, and they pinch off into micron-sized droplets which
are subsequently suspended in the continuous phase. These are among the widely used
micro emulsification apparatus. Meanwhile, the production of droplets with an expectable
and reproducible size distribution determines their possible uses (including the synthesis
of polymer colloids), numerous researchers have investigated different features of the
emulsification procedure. It was concluded that the controlling parameters of droplet sizes
include liquids’ properties, flow rates of the two immiscible phases, and the microfluidic
device design [20].
In this study, the dynamics of the break-up were discussed considering the model
of rate-of-flow-controlled-breakup (or squeezing). According to this model, the droplet
size was dependent on the flow rate ratio of the two dispersed and continuous phases.
Practically, our study indicates guidelines for generating droplets with a predetermined
size and polydispersity from liquids with well-defined macroscopic properties, utilizing a
MFFD. Additionally, significant biocompatibility and cell proliferation of alginate hydrogels
containing citrated magnetic NPs present the high potential of these synthetic materials
for novel applications such as 3D bioprinting of intrinsic magnetic scaffolds, injectable
granulated hydrogels, and synergistic and targeted cancer therapy using controlled drug
delivery and hyperthermia. This study provides a useful approach and comprehensive
guideline for obtaining complex biocompatible materials with active magnetic properties.

2. Results and Discussion

2.1. Properties of Magnetic NPs Synthesized by Co-Precipitation Method
2.1.1. Morphology and Particle Size
The FE-SEM image of magnetite NPs is shown in Figure 1a. According to Figure 1a,
the morphology of the synthesized magnetite NPs was spherical with the mean as-prepared
 2. Results and Discussion
2.1. Properties of Magnetic NPs Synthesized by Co‐Precipitation Method
Gels 2023, 9, 501 2.1.1. Morphology and Particle Size 3 of 14
The FE-SEM image of magnetite NPs is shown in Figure 1a. According to Figure 1a,
the morphology of the synthesized magnetite NPs was spherical with the mean as-pre-
pared nanoparticle
nanoparticle size of 29.1 ± 29.1
size of ± 2.5The
2.5 nm. nm. average
The average particle
particle sizesize
waswas obtained
obtained after
after meas-
measuring
uring the diameter of 50 NPs in
the diameter of 50 NPs in the image.the image.

80

60

Magnetization (emu g )
40

-1
20

-20

-40

-60

-80
-15,000 -10,000 -5,000 0 5,000 10,000 15,000
Applied field (Oe)

(a) (b)

(c) (d)
Figure 1. (a) FE-SEM image of magnetite NPs synthesized by the co-precipitation method, (b) M-H
Figure 1. (a) FE-SEM image of magnetite NPs synthesized by the co-precipitation method, (b) M-H
diagram of magnetite NPs synthesized by the co-precipitation method, (c) FT-IR spectra of triso-
diagram of magnetite NPs synthesized by the co-precipitation method, (c) FT-IR spectra of trisodium
dium citrate, magnetite NPs, and citrated magnetite NPs, and (d) hydrodynamic size of magnetite
citrate, magnetite
NPs before NPs,citration.
and after and citrated magnetite NPs, and (d) hydrodynamic size of magnetite NPs
before and after citration.
2.1.2. Magnetic Properties
2.1.2. Magnetic Properties
Magnetic measurements of NPs were performed using a vibrating sample magne-
Magnetic measurements of NPs were performed using a vibrating sample magnetome-
tometer (VSM, MagKav Co., Kashan, Iran) at room temperature. One of the special prop-
ter (VSM, MagKav Co., Kashan, Iran) at room temperature. One of the special properties of
erties of magnetic NPs is their superparamagnetic behavior. When a magnetic material
magnetic
dimensionNPsis is
intheir superparamagnetic
nanoscale, each magnetic behavior.
domain is When
locateda in
magnetic material
a separate dimension
NP. When the
is particle
in nanoscale, each magnetic domain is located in a separate NP. When the particle
size is less than 30 nm, they inhibit superparamagnetic behavior [21]. The VSM size is
less than 30 nm, they inhibit superparamagnetic behavior [21]. The VSM curve
curve of synthesized NPs at room temperature is shown in Figure 1b. As seen in Figure of synthe-
sized NPscoercivity
1b, the at room temperature
and remanenceis shown in Figure
are zero. 1b. As
In addition, seen
the in Figure
saturation 1b, the coercivity
magnetization is
and remanence
66.92 emu g .−1 are zero. In addition, the saturation magnetization is 66.92 emu g−1 .

2.1.3. FT-IR Investigation

The FT-IR spectra of samples were obtained using a Perkin-Elmer 781 nanoparticle
spectrophotometer (Waltham, MA, USA). In this study, the samples were formulated as tablets
in solid KBr solution and the spectrum was obtained in the mid-range of 4000–400 cm−1 .
The FT-IR of bare magnetic NPs, citrated magnetic NPs, and trisodium citrate as the
control group are shown in Figure 1c. Two main peaks of magnetite in the FT-IR spectrum
have appeared in the wavelength range of 550–650 cm−1 showing Fe–O groups. The peak
at 3450 cm−1 is due to the stretching vibrations of O-H groups [22]. According to the FT-IR
spectrum of citrated NPs, there is a new peak at 2352 cm−1 . The corresponding peak in
Gels 2023, 9, 501 4 of 14

the spectrum of trisodium citrate is 2250 cm−1 . This indicates that the citrate is attached to
the oxygen groups on the magnetite via the covalent bond and resulted in the C=O bond’s
shift from 2250 to 2352 cm−1 . This shift indicates the formation of a carboxyl bond between
sodium citrate and magnetite NPs [23].

2.1.4. Hydrodynamic Size Investigation

The hydrodynamic size distributions of NPs were taken using dynamic light scattering
(DLS, Malvern Zetasizer Nano ZS, Malvern, UK).
The DLS results of NPs before and after being citrated are shown in Figure 1d. The
hydrodynamic size before being citrated was 142 nm which reached 826.7 nm after being
citrated. Being citrated causes an increase of NP stability in the solution and prevents
sedimentation during the microfluidic process.

2.2. Micro Gel Production in the Microfluidic Chip

In this research, experiments were performed based on the flow-focusing geometry
for the formation of droplets in a continuous phase (oil phase) from an immiscible phase
(hydrogel precursors). This method enables us to produce high-speed and one-step hydro-
gels’ production in the desired dimensions. The microfluidic chip efficiency in microgels’
synthesis was imaged by a biological optical microscope (CX22 Olympus, Tokyo, Japan)
which was equipped with a recorder camera system.
Two different designs of microfluidic chips were used; the triple junction geometry
was the same for both designs. Droplet formation was investigated in both designs and
droplet formation in the triple junction in both chips was in the same condition of flow
rate ratio. In the chip with spiral channels, the size of collected droplets was not uniform
because the spiral design acts as a mixer, and in the channel path, they mix to etch other or
separate into smaller droplets (Figure 2a). To find the average size of hydrogels in an image,
approximately 100 hydrogels were measured and averaged by ImageJ software v1.53. The
average diameter of microgels is 38.7 (±10) µm. Droplets produced in the chips with
Gels 2023, 9, x FOR PEER REVIEWparallel channels approximately were not mixed or separated. They were collected 5 of at
15 the
triple junction point. Hence, in this study, the results of the droplets produced in the chip
with parallel channels were reported.

(a) (b)
Figure2.2.(a)
Figure (a) Microscopic
Microscopic image
imageofofcollected microgels
collected produced
microgels in theinspinal
produced chip, (b)
the spinal Optical
chip, mi-
(b) Optical
croscope image of triple junction in microfluidic chip.
microscope image of triple junction in microfluidic chip.

AAwide
widerange
rangeofofdroplet
droplet formation
formation patterns
patternswere
wereobserved
observedbased onon
based thethe
input rates
input rates
of two phases. The droplets’ size change was quantified by two parameters: the
of two phases. The droplets’ size change was quantified by two parameters: the oil phase oil phase
rate
rate (Q(Q o) and the ratio of the hydrophilic phase input rate to the oil phase rate (Qi/Qo). The
o ) and the ratio of the hydrophilic phase input rate to the oil phase rate (Qi /Qo ). The
oil phase rate has assumed as the sum of the input rates of two oil channels. The formation
oil phase rate has assumed as the sum of the input rates of two oil channels. The formation
of droplets was observed both uniformly and non-uniformly. The geometry of the utilized
of droplets was observed both uniformly and non-uniformly. The geometry of the utilized
microfluidic system is shown in Figure 2b, where wo is 160 µm, wi and w1 are 110 µm, and
w2 is 200 µm. The oil phase flows from the two side channels and the hydrophilic phase
flows from the middle channel. A shear force is applied from the two oil flows to the hy-
drophilic phase and disrupts it, and the droplets are formed.
In this research, the input rate of the oil phase (Qo) was always greater than the input
Gels 2023, 9, 501 5 of 14

microfluidic system is shown in Figure 2b, where wo is 160 µm, wi and w1 are 110 µm,
and w2 is 200 µm. The oil phase flows from the two side channels and the hydrophilic
phase flows from the middle channel. A shear force is applied from the two oil flows to the
hydrophilic phase and disrupts it, and the droplets are formed.
In this research, the input rate of the oil phase (Qo ) was always greater than the input
rate of the hydrophilic phase (Qi ). Two ratios of Qi /Qo = 1/5 and 1/20 were selected and
studied. The oil phase rate has assumed as the sum of the input rates of two oil channels
and was tuned from 0.2 to 20 mL h−1 . Droplet formation was observed and recorded by
the optical microscope and the connected camera.
After the completion of numerous experiments for the formation of droplets in the
microfluidic system, the following table was achieved for various cases.
Figure 3 shows different modes of droplet production in the microfluidic system.
According to Figure 3, in the case of Qo = 2 mL h−1 and Qi /Qo = 1/20, the formation of
droplets is along with the formation of some satellites (Figure 3d), which indicates a non-
uniform distribution of droplets’ sizes. In the case of Qo = 0.2 mL h−1 and Qi /Qo = 1/20,
Gels 2023, 9, x FOR PEER REVIEW
the formation of droplets with more homogeneity in size distribution can be seen6 of(Figure
15
3f).
The droplet separation occurs in the place of channel narrowing.

Figure 3. Different modes of droplet production in the microfluidic system, regarding the physical
Figure 3. Different modes of droplet production in the microfluidic system, regarding the physical
properties of phases that are constant in this study jetting mode (a, b), dripping mode (c, e, f) and
properties of phases that
satellite are constant
formation in this study jetting mode (a,b), dripping mode (c,e,f) and
(d) were observed.
satellite formation (d) were observed.
According to Figure 3c,e,f, a uniform size distribution of the droplets can be ob-
served. Furthermore, in some cases (Qo = 20 mL h−1 and Qi/Qo = 1/5) the droplets merged
Accordingandto Figure 3c,e,f, a uniform size distribution of the droplets can be observed.
formed larger droplets when they were crossing through the channels and subse-
Furthermore, in someincases (Qo =(Figure
20 mL −1 and Q /Q = 1/5) the droplets merged and
quently, the pathway 3a).hRegarding the
i physical
o properties of phases that are
formed larger droplets when they were crossing through
constant in this study, two different modes thedripping
occurred: the channels and subsequently,
and jetting created by in
changing the two phases’ flow rates. In the case of dripping, the middle phase flow is
interrupted in the narrowing place of the channel or near it and the droplets are formed.
(Figure 3c,e,f). In the jetting mode, the middle phase extends several hundreds of microm-
eters downstream of the narrowing section, and may (droplet formation in Figure 3a,b) or
may not (fiber formation) be interrupted.
Gels 2023, 9, 501 6 of 14

the pathway (Figure 3a). Regarding the physical properties of phases that are constant in
this study, two different modes occurred: the dripping and jetting created by changing the
two phases’ flow rates. In the case of dripping, the middle phase flow is interrupted in the
narrowing place of the channel or near it and the droplets are formed. (Figure 3c,e,f). In
the jetting mode, the middle phase extends several hundreds of micrometers downstream
of the narrowing section, and may (droplet formation in Figure 3a,b) or may not (fiber
formation) be interrupted.
With constant Qi /Qo and small values of Qi and Qo (0.2 mL h−1 ), the dripping mode
occurs and larger droplets are formed (Figure 3e with a diameter of about 120 µm).
In moderate values of Qi and Qo (2 mL h−1 ), the dripping mode occurs again; the droplet
size distribution is more uniform and smaller droplets are formed rather than low velocities
(Figure 3c with a diameter of about 75 µm). In a narrow range of moderate values of Qi and
Qo , small satellite droplets were formed with a non-uniform size distribution (Figure 3a).
At high values of Qi and Qo (20 mL h−1 ), the jet stream occurs and the middle
phase extends several hundreds of micrometers downstream of the narrowing section
(Figure 3a,b). In tests carried out during the transition between the dripping and jetting
mode, the tip of the middle phase extends abruptly and was unstable.
The droplets produced in this case were initially fine but they merged with each other
and larger droplets formed, so the size distribution became non-uniform [20]. In Figure 3a,
the coagulation of droplets in the channel can be seen.

2.3. Characterization of Microgels Containing Magnetic NPs

Microscopic images of hydrogels were obtained by fluorescence microscope (Olympus
BX51, Japan) in transmission mode without any fluorescent labeling in order to ensure their
structures and morphologies were not disrupted. The size of about 100 hydrogels in each
photo was measured by ImageJ software and averaged to obtain hydrogel average diameters.
Figure 4a shows microgels with non-uniform sizes with an average diameter of
25.64 µm resulting from the middle-phase jet flow. In Figure 4b, where the middle phase
flow rate is reduced, the average diameter of the microgels is reduced to 19.5 µm and the
size distribution is nearly uniform. The heterogeneity in droplet sizes was analyzed by
a developed model by Higgins et al. [24]. According to their model, the effect of hetero-
geneity was quantified by the degree of inconsistency in the studies’ results (I2 value, the
percentage of observed total variation across studies, which is due to real heterogeneity
rather than chance). The heterogeneity in the size of microgels in Figure 4a was calculated
at 71%, while it was 21% for microgels in Figure 4b; knowing that the value of 0% indicates
no observed heterogeneity and larger values show increasing heterogeneity.
Figure 5a shows the VSM result of alginate microgels containing magnetite NPs.
According to Figure 5a, coercivity and remanence are zero, therefore the encapsulated NPs
in hydrogels preserve the superparamagnetic property. The saturation magnetization is
1.38 emu g−1 . The reduction of saturation magnetization in hydrogels containing magnetic
NPs (in comparison with magnetite) can be due to non-crystalline structures’ attachment
on magnetic NPs. Any crystalline disorder in the surface of NPs can increase surface
anisotropy and significantly reduce saturation magnetization [25]. In addition, the weight
percentage of MNPs in microgels containing magnetic NPs decreases, and the diamagnetic
hydrogel surrounds the Fe3 O4 ; both lead to a decrease in magnetic properties [26].
The FT-IR spectrum of alginate hydrogels with and without magnetite NPs is shown
in Figure 5b. In order to have a better comparison, the spectrum of sodium alginate is
also taken. In the sodium alginate spectrum, the main peaks that appeared at 3374, 1610,
1415, and 1036 cm−1 were related to OH, COO (asymmetric stretching vibrations), COO
(symmetric stretching vibration), and COC bonds, respectively.
These peaks in the alginate microgels spectrum appeared at 3390, 1600, 1415, and
1037 cm−1 for OH, COO (asymmetric stretching vibrations), COO (symmetric stretching
vibration), and COC bonds, respectively [27,28]. There is a small shift in peaks’ locations
in microgels. The reason for this shift can be due to the structure folding of the molecules
 At high values of Qi and Qo (20 mL h−1), the jet stream occurs and the middle phase
extends several hundreds of micrometers downstream of the narrowing section (Figure
3a,b). In tests carried out during the transition between the dripping and jetting mode, the
tip of the middle phase extends abruptly and was unstable.
The droplets produced in this case were initially fine but they merged with each other
Gels 2023, 9, 501 and larger droplets formed, so the size distribution became non-uniform [20]. In Figure 7 of 14
3a, the coagulation of droplets in the channel can be seen.

2.3. Characterization of Microgels Containing Magnetic NPs

and ionicMicroscopic
bonds’ formation
images ofbetween
hydrogelscarboxylate
were obtained groups and calcium
by fluorescence ions. Furthermore,
microscope (Olym- no
newpuspeakBX51, Japan) in transmission
formation indicates no mode
new without any fluorescent
covalent labeling in
bond formation order to ensure
between the comprising
their structures
hydrogel molecules.and morphologies were not disrupted.
Figure 5c schematically The size the
describes of about 100 hydrogels
mechanism in egg box
of the
each photo was measured by ImageJ software and averaged to obtain hydrogel average
structure formation and ionic cross-linking of the alginate chains. Egg box structure refers
diameters.
to the formation of divalent
Figure 4a shows metal
microgels withions, such assizes
non-uniform calcium or average
with an magnesium,
diametercross-linking
of 25.64 with
carboxylate groups in alginate. This results in a 3D network of chains
µm resulting from the middle-phase jet flow. In Figure 4b, where the middle phase flowthat provides stability
to the alginate
rate matrix
is reduced, [29]. The
the average strength
diameter ofmicrogels
of the hydrogels could be
is reduced ascribed
to 19.5 µm andto the
thesize
crosslinking
distribution
ability of calciumis nearly
ions.uniform.
D’ElíaThe heterogeneity
et al. in droplet
[30] explained thatsizes was analyzed
hydrogels with by a de- levels of
greater
Ca2+veloped
tend to model
havebyhigher
Higginsstorage
et al. [24]. According(energy
modulus to their model,
storedtheineffect of heterogeneity
the elastic structure) values
was quantified by the degree of inconsistency in the studies’ results (I2 value, the percent-
in the linear viscoelastic range which results in higher strength. Moreover, this structure
age of observed total variation across studies, which is due to real heterogeneity rather
reduces the water content within the gel, which contributes to its stability by limiting the
than chance). The heterogeneity in the size of microgels in Figure 4a was calculated at
mobility of the
71%, while polymer
it was 21% forchains
microgelsandin preventing the chains
Figure 4b; knowing from
that the valuebecoming soluble in an
of 0% indicates
aqueous solution [31].
no observed heterogeneity and larger values show increasing heterogeneity.

Gels 2023, 9, x FOR PEER REVIEW 8 of 15

NPs (in comparison with magnetite) can be due to non-crystalline structures’ attachment
on magnetic NPs. Any crystalline disorder in the surface of NPs can increase surface ani-
Figure
sotropy4. Microscopy image (inreduce
and significantly transmission mode)magnetization
saturation of: (a) non-uniform distribution
[25]. of droplet
In addition, sizes
the weight
Figure
and4.(b)Microscopy
near uniformimage (in transmission
distribution of droplet mode) of: (a) non-uniform
sizes. distribution of droplet sizes
percentage of MNPs in microgels containing magnetic NPs decreases, and the diamag-
and (b) near uniform distribution of droplet sizes.
netic hydrogel surrounds the Fe3O4; both lead to a decrease in magnetic properties [26].
Figure 5a shows the VSM result of alginate microgels containing magnetite NPs. Ac-
cording to Figure 5a, coercivity and remanence are zero, therefore the encapsulated NPs
1.5 in hydrogels preserve the superparamagnetic property. The saturation magnetization is
1.38 emu g−1. The reduction of saturation magnetization in hydrogels containing magnetic
1.0
Magnetization (emu g-1)

0.5

0.0

-0.5

-1.0

-1.5

-15,000 -10,000 -5,000 0 5,000 10,000 15,000

Applied field (Oe)
(a) (b)

(c)
Figure 5. (a) M-H diagram of alginate microgels containing magnetite NPs, (b) FT-IR spectra of
Figure 5. (a) M-H diagram of alginate microgels containing magnetite NPs, (b) FT-IR spectra of
sodium alginate (S.Alg), alginate microgels (Alg.M.) and alginate microgels containing citrated
sodium alginate
magnetite NPs(S.Alg), alginate and
(Alg.MNP-Cit.M), microgels
(c) ionic (Alg.M.) and alginate
bonding interaction microgels
between containing citrated
Ca2+ and carboxylate
magnetite NPs (Alg.MNP-Cit.M), and (c) ionic bonding interaction between Ca2+ and carboxylate
groups in alginate hydrogels.
groups in alginate hydrogels.
The FT-IR spectrum of alginate hydrogels with and without magnetite NPs is shown
in Figure 5b. In order to have a better comparison, the spectrum of sodium alginate is also
taken. In the sodium alginate spectrum, the main peaks that appeared at 3374, 1610, 1415,
and 1036 cm−1 were related to OH, COO (asymmetric stretching vibrations), COO (sym-
metric stretching vibration), and COC bonds, respectively.
These peaks in the alginate microgels spectrum appeared at 3390, 1600, 1415, and
Gels 2023, 9, 501 8 of 14

In the alginate microgels and citrated magnetite NPs spectrums, the formation of a
bond between citrate and magnetite NPs (C=O) appeared at 2356 cm−1 [23,32].
The slight shift of this peak also indicates the weak bonds between alginate and citrate
on the surface of NPs. The peaks appeared at 570, 1415, 1612, 1037, and 3405 cm−1 which
are due to Fe-O, COO (symmetric stretching vibrations), COO (asymmetric stretching
vibration), COC, and OH, respectively [33–35]. This result proves the presence of magnetite
NPs inside the hydrogels.

2.4. Biocompatibility of Magnetite NPs before and after Citration

The cytotoxicity results of magnetite NPs before and after citration at different concen-
trations are shown in Figure 6a. The significance of differences between the biocompatibility
of magnetite NPs and citrated magnetite NPs at different concentrations within the control
group was statistically analyzed by SPSS software. The group statistics of the measurement
results were analyzed using ANOVA post-hoc Tukey. The value of p < 0.05 was defined
as a condition of significant differences. If the significant difference parameter (Sig) is less
than 0.05, the difference is significant and means a decrease in cell growth compared to
the control group. At 200 µg µL−1 and lower concentrations, the cytotoxicity of both bare
magnetite NPs and citrated magnetite NPs were not significant. However, in the citrated
NPs, at the concentration of 200 µg µL−1 , the rate of stem cell growth was significantly
higher than similar concentrations of non-citrated NPs (p = 0.009634). Proper cell growth in
the presence of citrated magnetite NPs indicates a decrease in cytotoxicity and an increase
in their biocompatibility through the citration process.
Cellular biocompatibility resulting from microgels (with and without magnetite NPs)
and their constituents prior to gelation for 24 and 72 h of cell proliferation is shown in
Figure 6b,c. According to statistical analysis by SPSS v29 software, the cell growth rate in
alginate microgels compared to alginate solution after 24 h of cell culture has a significant
parameter of 0.002. Given this low p value, gelation has increased the bioactivity of the
initial solutions. Similar to hydrogels without citrated magnetite NPs, there are similar
conditions in microgels containing NPs before and after gelation. Cell proliferation in
alginate microgels containing NPs during the first 24 h of culture was significantly more
than that of the hydrogels’ precursor.
After one day only, alginate nanoparticles containing citrated magnetite nanoparticles
had no significant difference with the control group and there was no decrease in cell
growth and proliferation in this group.
The cell growth rate in alginate microgels compared to alginate solution after 72 h
of cell culture has a significant parameter of 0.017, then sodium alginate microgels have
better cell proliferation than sodium alginate. Cell proliferation in alginate microgels
containing citrated magnetite NPs was significantly higher than net alginate microgels
within 72 h post-culture (p = 0.042). It could be due to how citrate groups (Ca2+ chelator)
induce a structural switch in a tight alginate network and produce a more permissive
microenvironment with a more open network [36]. This type of switch affects not only the
structural features (i.e., the mesh size) but also the mechanical properties of hydrogels. In
addition, diffusion of biological compounds and cell motility may be affected by alteration
in the network mesh size [37].
The significant difference in cell proliferation in all groups after 72 h was compared by
pairwise comparison. After three days, all groups except for the sodium alginate group
had no significant difference and were able to have good cell proliferation.
 (Sig) is less than 0.05, the difference is significant and means a decrease in cell growth
compared to the control group. At 200 µg µL−1 and lower concentrations, the cytotoxicity
of both bare magnetite NPs and citrated magnetite NPs were not significant. However, in
the citrated NPs, at the concentration of 200 µg µL−1, the rate of stem cell growth was
significantly higher than similar concentrations of non-citrated NPs (p = 0.009634). Proper
Gels 2023, 9, 501 9 of 14
cell growth in the presence of citrated magnetite NPs indicates a decrease in cytotoxicity
and an increase in their biocompatibility through the citration process.

Figure 6. (a) Magnetite NPs biocompatibility before and after citration at different concentrations in
the presence of human mesenchymal stem cells after 24 h (*: p < 0.05, **: p < 0.001); (b) biocompatibility
test of alginate based microgels and their constituents after 1 day (*: p < 0.05 in comparison with
control group); and (c) biocompatibility test of alginate based microgels and their constituents after
3 days. (*: p < 0.05).

3. Conclusions
In this study, alginate microgels containing citrated magnetite NPs were produced
by the microfluidic flow-focusing device. Superparamagnetic NPs with an average size
of 29.1 ± 2.5 nm and saturation magnetization of 66.92 emu g−1 were synthesized via the
co-precipitation method. The citration process was performed on NPs in order to prevent
MNPs’ agglomeration. DLS results indicated an increase in MNPs hydrodynamic diameter
from 142 nm to 826.7 nm after citration so dispersion and stability in the aqueous phase was
improved. In addition, cell culture analysis showed significantly higher biocompatibility of
citrated MNPs in comparison with non-citrated MNPs in the concentration of 200 µg µL−1 .
The alginate droplets were produced in the MFFD and the size and polydispersity of
droplets were discussed in relation to the flow rate ratio of two immiscible phases of oil and
hydrogel precursor based on the model of rate-of-flow-controlled-breakup (or squeezing).
FT-IR results stated the chemical attachment of citrate groups on MNPs and the existence
of MNPs in the hydrogels. The values of coercivity and remanence remained at zero after
encapsulation of citrated MNPs in alginate hydrogels but the saturation magnetization
decreased to 1.38 emu g−1 due to non-crystalline structures attachment on magnetic NPs.
Cell proliferation in alginate microgels containing citrated magnetite NPs was significantly
higher than net alginate microgels within 72 h post-culture (p = 0.042). Moreover, at
200 µg µL−1 and lower concentrations, the cytotoxicity of magnetite NPs and citrated
magnetite NPs were not significant. In brief, the results indicated that alginate hydrogels
containing citrated MNPs produced in this work showed significant biocompatibility. On
Gels 2023, 9, 501 10 of 14

the other hand, preserving the superparamagnetic properties of MNPs after trapping
them in hydrogel makes this multifunctional material suitable for magnetic resonance
imaging (MRI), hyperthermia, magnetic active targeted drug delivery, tissue engineering
scaffolds, and 3D cell culture applications. Furthermore, the predetermined size and
polydispersity with well-defined macroscopic properties, utilizing a MFFD achieved in
this work, would be an important step to progress using injectable granulated hydrogels in
regenerative medicine.

4. Materials and Methods

4.1. Materials
Poly(dimethylsiloxane) and SYLGARD184 silicone elastomer kit were from Dow
Corning. Soybean oil, sodium alginate, calcium acetate, sodium citrate (citric acid trisodium
salt dehydrate), span 80, resazurin sodium salt, phosphate-buffered saline (PBS), penicillin,
and streptomycin were purchased from Sigma-Aldrich company (Washington, DC, USA).
FeCl3 , FeCl2 .4H2 O, and NH4 OH were purchased from Merck company (Darmstadt,
Germany). Minimum essential medium alpha (MEM α) and trypan blue solution 0.4% were
bought from Fisher Scientific (Portsmouth, NH, USA). Fetal bovine serum (FBS) and trypsin
were purchased from Life Technologies company (Carlsbad, CA, USA). More details on
materials specifications are listed in Table 1.

Table 1. Specifications of materials used in this study.

Material Company CAS No.

FeCl3 Merck 7705-08-0
FeCl2 .4H2 O Merck 13478-10-9
NH4 OH Merck 1336-21-6
Ethanol Merck 64-17-5
Acetone Merck 67-64-1
Sodium citrate Sigma Aldrich 6132-04-3
PDMS Sigma Aldrich 9016-00-6
Sodium alginate Sigma Aldrich 9005-38-3
Calcium acetate Sigma Aldrich 114460-21-8
Soybean oil Sigma Aldrich 8001-22-7
Span 80 Sigma Aldrich 1338-43-8

4.2. Methods
4.2.1. Magnetite Nanoparticles Synthesis
The co-precipitation process was used for the synthesis of Fe3 O4 magnetic NPs. For this
purpose, 150 mL of deionized water containing 78 mM of FeCl3 and 150 mL of deionized
water containing 39 mM of FeCl2 .4H2 O were prepared separately. The solutions were
magnetically stirred and then mixed in a tree neck balloon under the nitrogen atmosphere.
The solution was then heated up to 80 ◦ C and 10 mL of 25% NH4 OH was added to the
stirring mixture dropwise until pH reached 11. The precipitates were then separated by
the centrifuge and washed one time with the water and centrifuged again to remove the
reaction by-products and finally were dried at room temperature.

4.2.2. 3D-Printing of the Mold

Two different designs of molds (Figure 7) were printed by EnvisionTEC with the
dimension accuracy of 25 µm by 84 mm lens and PIC 100 resin. The prepared models
were washed with ethanol and then immersed in an ethanol bath for 20 min to remove the
remaining resin from the surface. After drying, the models were exposed to a visible light
 Gels 2023, 9, 501 11 of 14
023, 9, x FOR PEER REVIEW 12 of 15

Gels 2023, 9, x FOR PEER REVIEW 12 of 15

flasher to cure the resin completely to prevent any crack formation in the next step when
molding by PDMS inmolding
the oven.byThe samples
PDMS wereoven.
in the exposed
Thetosamples
1000 flashes.
wereFor this pur-to 1000 flashes. For this purpose,
exposed
pose, a flasher device with the frequency of 1 flash per second was used. Finally, the sam-
a flasher device
molding with in
by PDMS the
thefrequency of 1 flash
oven. The samples wereper second
exposed was flashes.
to 1000 used. Finally, the samples
For this pur-
ples were washed with ethanol and deionized water in order to remove any more pollu-
were pose, a flasher
washed withdevice with the
ethanol andfrequency of 1 water
deionized flash perinsecond
order was
to used. Finally,
remove any the sam-pollution
more
tion from the surface of the samples.
fromples
the were washed
surface with
of the ethanol and deionized water in order to remove any more pollu-
samples.
tion from the surface of the samples.

(a)
(a)

(b)
Figure 7. 3D view of designed
Figure models
7. 3D for microfluidic
view chip mold
of designed with:for
models (a) microfluidic
spinal
(b) channel chip
and (b)
mold with: (a) spinal channel and
parallel channels.
(b) parallel channels.
Figure 7. 3D view of designed models for microfluidic chip mold with: (a) spinal channel and (b)
parallel channels.
4.2.3. Synthesize of Citrated Magnetite Nanoparticles
4.2.3. Synthesize of Citrated Magnetite Nanoparticles
For this purpose, 1.5 4.2.3.
mg ofSynthesize
synthesized magnetite
of Citrated NPs were
Magnetite dispersed in 2 mL of
Nanoparticles
For this
deionized water in an ultrasonic purpose,
bath 1.5 mg
for 2 h (Figure 8a).ofThen,
synthesized magnetite
1.5 mg of sodium NPs were dispersed in 2 mL of
citrate
deionized Forwater
this purpose, 1.5 mg of synthesized magnetite
h (FigureNPs were dispersed in 2 mL of
was added to the mixture and heated toin
80 an
°C ultrasonic bath for
for 60 min (Figure 8b).2Immediately 8a).
afterThen, 1.5 mg of sodium citrate
deionized water in an ultrasonic bath for 2 h (Figure◦ 8a). Then, 1.5 mg of sodium citrate
removing the mixturewasfromadded to the
the oven, 2 mLmixture and
of acetone heated
were addedto 80theCresultant
and for 60 min
mix- (Figure 8b). Immediately after
was added to the mixture and heated to 80 °C for 60 min (Figure 8b). Immediately after
ture was stored at room temperature
removing the overnight. from
mixture Duringthethisoven,
time, the
2 citrated
mL of NPs werewere added and the resultant
acetone
removing the mixture from the oven, 2 mL of acetone were added and the resultant mix-
precipitated and themixture
upper solution was separable (Figure 8c,d). After sucking theDuring
top
ture was stored
was stored at at room
room temperature
temperature overnight.
overnight. During this time, thethis time,NPs
citrated the were
citrated NPs
solution, the sediments were separated.
wereprecipitated
precipitated and
and thethe upper
upper solution
solution was was separable
separable (Figure(Figure 8c,d).
8c,d). After After the
sucking sucking
top the top
solution, the sediments were
solution, the sediments were separated.separated.

Figure 8. Citration steps of Fe3O4 NPs: (a) after the addition of deionized water to the magnetic NPs
and dispersion in an ultrasonic bath, (b) after the addition of sodium citrate and placing it in the
Figure 8. Citration steps of Fe3O4 NPs: (a) after the addition of deionized water to the magnetic NPs
Figureand Citration in
8. dispersion steps of Fe3 O4bath,
an ultrasonic NPs:(b)(a) after
after thethe addition
addition of deionized
of sodium water
citrate and to the
placing it inmagnetic
the NPs
and dispersion in an ultrasonic bath, (b) after the addition of sodium citrate and placing it in the oven,
(c) after the addition of acetone and precipitation of citrated magnetic NPs, and (d) the attraction of
citrated magnetic NPs to the magnet.

4.2.4. Production of Microfluidic Chip

PDMS and the curing agent were mixed in at a ratio of 1:10 wt% in order of the
microfluidic chip molding and then placed in the vacuum chamber for 30 min to be
Gels 2023, 9, 501 12 of 14

degassed. The vacuum was removed and applied 2–3 times. The models remained for
45 min in a 75 ◦ C oven for PDMS curing. In the next step, the punched PDMS replica and
bare PDMS plate surface were activated by plasma spray and coupled together. The chips
remained for 12 h in a 115 ◦ C oven to achieve a stronger bond.

4.2.5. Solutions Preparation

A total of two hydrophilic and hydrophobic phases were prepared for the synthesis of
microgel droplets in the chip.
To prepare for the hydrophilic dispersed phase, 1 wt% of sodium alginate aqueous
solution was prepared by adding a certain amount of sodium alginate salt to DI water and
it was maintained for 2 h in an incubator on rotating axels. The solution passed through a
450 nm filter to remove any unsolved particles and dust. A total of 500 µL of deionized
water were added to 1.5 mg of citrated magnetite NPs and pipetaged for several times. The
solution was added to 2 mL of a 1 wt% of sodium alginate aqueous solution and mixed
using a vortex mixer.
To prepare for the hydrophobic continuous phase, a solution containing 2 wt% of
calcium acetate and 3 wt% of Span 80 surfactant in soybean oil were prepared and vortexed
for 30 min and then mixed for 12 h by magnetic stirring. The organic solution was also
filtered by a 450 nm filter to remove any unwanted particles. A collection reservoir solution
containing 3 wt% of Span 80 surfactant in soybean oil was prepared and magnetically
stirred for 30 min. This solution was used for saving the hydrogel droplets outgoing from
the microfluidic chip.

4.2.6. Droplet Formation

Different input rates were tested to investigate the possibility of the formation of a
stable droplet in the chip. The examined rate ranges (sum of two rates from two channels)
were 30–0.2 mL and 3–0.01 mL per hour for the hydrophobic and hydrophilic phases,
respectively. It should be noted that flow rates higher than 30 mL h−1 resulted in chip
failure because the PDMS attachment to glass was not strong enough to overcome those
flow rates. Finally, the achieved droplets were stored in 3 wt% of Span 80 in Soybean oil.

4.2.7. Cell Culture

Human mesenchymal stem cells (hMSCs) were used in this study. The cell line was
obtained from the national cell bank (Pasteur Institute of Iran, Tehran, Iran). The hMSCs
were proliferated in α MEM medium supplemented with 10% of FBS and 1% of penicillin,
and 1% of streptomycin in 125 cm2 flasks at 37 ◦ C in a sterile incubator. Cell counting was
performed by an automated cell counter, which was used to evaluate cytotoxicity.

4.2.8. Alamar Blue Cell Proliferation Test

Due to the high potential of the produced hydrogels as an MRI contrast agent and as
bio-ink for 3D printing of scaffolds and injectable hydrogels, the toxicity test was performed.
For this purpose, the alamar blue method was used for a cell proliferation assay with NPs
and hydrogels. The NPs and hydrogels were exposed to UV radiation for 40 min prior to
cell testing to eliminate possible microbial contamination.
To investigate the toxicity or cell proliferation of NPs and hydrogels, 10,000 cells were
plated on each of the 96-well plates. Approximately 200 µL of α MEM medium containing
10% of FBS and 1% of antibiotic were added to each well. Plates were incubated at 37 ◦ C
for 24 h. The amount of CO2 incubated in cell culture was adjusted to 5%. After one day,
the cells were stuck to the bottom of the plate. The cell culture medium was evacuated
and replaced with a culture medium containing a certain amount of NPs or hydrogels. To
investigate the cell proliferation potential of the specimens, hydrogels and NPs were placed
into the wells at the bottom of the 96-well plates, then 200 µL of culture medium containing
10,000 cells was poured onto them. Cellular proliferation was determined after a specific
time by the alamar blue assay. The procedure was performed by adding 20 µL of alamar
Gels 2023, 9, 501 13 of 14

blue’s solution containing 40 mM of resazurin sodium salt in PBS medium to each well.
Then it was incubated in the dark for 4 h at 37 ◦ C. A total of 100 µL of the top solution
of each well was transferred to a new plate. The absorbance of each well, indicating cell
viability, was measured by the Elisa Plate Reader (Thermo Scientific, Waltham, MA, USA)
at 570 nm. At the same time, the absorbance was measured at 630 nm to eliminate the effect
of the background on absorption. Each process was repeated three times. In addition, wells
containing cells without NPs and hydrogels were selected as the control group.

Author Contributions: Conceptualization, S.A.S.E.; Data curation, F.M.; Formal analysis, F.M.;
Investigation, F.M. and Z.L.; Methodology, F.M. and V.Z.; Project administration, S.A.S.E.; Resources,
S.A.S.E. and B.H.; Supervision, S.A.S.E.; Validation, F.M. and V.Z.; Writing—original draft, F.M.;
Writing—review & editing, Z.L. and B.H. All authors have read and agreed to the published version
of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
first author.
Acknowledgments: The authors would like to thank Jos Malda, Harrie Weinans, Yang Li, and Saber
Amin Yavari for providing access to the Utrecht biofabrication facility, the introduction of DLP 3D
printer to prepare PDMS chips, as well as the microfluidic setup to prepare microgels.
Conflicts of Interest: The authors declare no conflict of interest.

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