Journal Pre-proof

Advances in biodegradable and injectable hydrogels for

biomedical applications

Yi Li, Hong Yu Yang, Doo Sung Lee

PII: S0168-3659(20)30731-8
DOI: https://doi.org/10.1016/j.jconrel.2020.12.008
Reference: COREL 10705

To appear in: Journal of Controlled Release

Received date: 26 August 2020

Revised date: 29 November 2020
Accepted date: 7 December 2020

Please cite this article as: Y. Li, H.Y. Yang and D.S. Lee, Advances in biodegradable and
injectable hydrogels for biomedical applications, Journal of Controlled Release (2020),
https://doi.org/10.1016/j.jconrel.2020.12.008

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© 2020 Published by Elsevier.

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Advances in biodegradable and injectable hydrogels for biomedical

applications
Yi Lia,b,* liyi@zjxu.edu.cn, Hong Yu Yangc, Doo Sung Leed,* dslee@skku.edu
a
College of Materials and Textile Engineering, Jiaxing University, Jiaxing 314001, Zhejiang, PR
China
b
Nanotechnology Research Institute (NRI), Jiaxing University, Jiaxing 314001, Zhejiang, PR

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China

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c
College of Materials Science and Engineering, Jilin Institute of Chemical Technology, Jilin City
132022, PR China
d
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Theranostic Macromolecules Research Center and School of Chemical Engineering,
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Sungkyunkwan University, Suwon, Gyeonggi-do 16419, Republic of Korea
*
Corresponding authors.
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Abstract
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In situ-forming injectable hydrogels are smart biomaterials that can be implanted into living
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bodies with minimal invasion. Due to pioneer work of Prof. Sung Wan Kim in this field,
injectable hydrogels have shown great potentials in many different biomedical applications.
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Biodegradable and injectable hydrogels can be administered at room temperature as viscous

polymer sols. They will degrade after accomplishing their tasks. Before injecting into living
bodies, active substances can be loaded into viscous polymer sols with a high loading efficiency
by simple mixing. After injecting into living bodies, active substances-loaded hydrogels can be
formed and active substances can be released in a controlled manner upon diffusion or polymer
degradation. Due to their outstanding properties and unique features, injectable hydrogels are
very promising in many biomedical applications including drug/protein/gene delivery, tissue
engineering, and regenerative medicine. In this review, we briefly introduce recent development
of several important types of in situ-forming injectable hydrogels reported by our group during
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the last three years. Important properties and potential applications (such as cancer therapy,
insulin release and wound healing) of these injectable hydrogels are reviewed. Challenges and
perspectives in this research field are also discussed.

Keywords: Injectable hydrogels; biodegradable polymers; environment-responsive; biomedical

applications

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1. Introduction

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During the last decade, hydrogels have been investigated extensively by researchers all over

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the world due to their outstanding physical/chemical/biological features and potential
applications in many different fields [1-4]. Hydrogels are generally soft and semi-solid matters
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that possess a three-dimensional polymer network. Polymers are crosslinked together. They act
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as a framework to support the structure of hydrogels. Pores in polymer networks allow hydrogels
to preserve a large amount of water or aqueous solutions [5, 6]. Owing to these unique features
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and properties, hydrogels are advantageous to be used as biomaterials in many biomedical

applications including drug delivery, tissue engineering, and regenerative medicine [7-11]. The
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presence of water or aqueous solutions endows hydrogels the ability to carry hydrophilic
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therapeutic agents and release them in a sustained manner for treating diseases or mimicking
structures of extracellular matrix (ECM) and tissues. Despite these unique properties,
applications of traditional preformed hydrogels are limited since these preformed hydrogels have
to be implanted subcutaneously which causes a lot of inconvenience to patients. To solve this
problem, in situ-forming and injectable hydrogels that undergo a sol-to-gel transition before and
after injection into the body have been developed [12, 13]. Injectable hydrogels are in a flowable
sol state (viscous polymer solution) before administration. They are ready to be injected into
living bodies using a syringe. Upon injection, the polymer solution can transform to a
non-flowable gel state in response to the body condition at the injection site. By pre-mixing in a
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sol state, functional agents can be co-injected into living bodies and encapsulated in in
situ-forming hydrogels for various purposes [14-16]. Injectability greatly improves the
convenience of administration of hydrogels while reducing worries of patients. To be
successfully used as injectable hydrogels, the viscous polymer solution should have relatively
low viscosity for easy injection. Gels should be formed in a short time to prevent undesired
leakage of loaded agents or moving to undesired sites. Biocompatibility of polymers and
contents of polymers in hydrogels should also be considered.

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Thermosensitive hydrogels are the most important and most widely investigated injectable

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hydrogels. These hydrogels undergo sol-to-gel transitions in response to temperature changes.

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Basic concepts, mechanisms and properties of these thermosensitive hydrogels have been
introduced and summarized in several important review articles [17-20]. In 1997, Prof. Sung
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Wan Kim and coworkers reported novel thermosensitive injectable hydrogels based on block
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copolymers consisting of poly(ethylene oxide) (PEO) and poly(L-lactic acid) (PLLA) [21].
Unlike traditional thermosensitive hydrogels based on poly(N-isopropylacrylamide) (PNIPAM)
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and PEO-b-poly(propylene oxide)-b-PEO (PEO-b-PPO-b-PEO), these PLLA-based

thermosensitive injectable hydrogels are biodegradable and biocompatible. Their work is
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considered a milestone in the development of injectable hydrogels for biomedical applications. It

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has inspired many researchers to work in this field. However, hydrogels reported in their work
can only form gels upon decreasing temperatures from elevated temperatures to body
temperature (37°C) which might cause loss of bioactivity when loading bioactive agents. In
follow-up works of Prof. Sung Wan Kim's group, aqueous solutions of triblock copolymers
consisting of PEO/poly(lactic-co-glycolic acid) (PLGA) and PEG/PCL that can form gels upon
increasing temperature from low temperature (or even room temperature) to body temperature
have been reported [22-25]. These triblock copolymers-based injectable hydrogels have been
successfully employed by Prof. Sung Wan Kim as depots for controlled release of insulin [26].
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These pioneering works have led research of injectable hydrogels to a new era and accelerated
applications of injectable hydrogels in biomedical fields.
To date, injectable hydrogels have been prepared by several different mechanisms, including
physical crosslinking, chemical crosslinking, and enzymatic crosslinking. Each type of injectable
hydrogels has their own advantages for different purposes. From the point of view of materials,
injectable hydrogels have been prepared using synthetic polymers, naturally occurring polymers,
and polymer/inorganic composites as constructing materials. In this review, we will briefly

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introduce recent development of several important types of in situ-forming injectable hydrogels

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reported by our group during the last three years. Potential applications of these injectable

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hydrogels with challenges and perspectives in this research field are discussed.
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2. Natural polymers-incorporated and temperature-sensitive injectable hydrogels
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For preparation of injectable hydrogels, poly(ε-caprolactone-co-lactide)-b-poly-(ethylene

glycol)-b-poly(ε-caprolactone-co-lactide) (PCLA-b-PEG-b-PCLA), an amphiphilic triblock
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copolymer, is a widely utilized biodegradable and temperature-sensitive polymer [27-29]. Gel

properties of PCLA-b-PEG-b-PCLA-based hydrogels are tunable by controlling chemical
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compositions or concentrations of the triblock copolymer within a wide range [30, 31]. To
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further extend applications of PCLA-b-PEG-b-PCLA hydrogels, our group has tried to

incorporate functional and naturally occurring polymers with PCLA-b-PEG-b-PCLA copolymer.
Injectable hydrogels formed from these new polymers have shown good potentials in several
different applications. For example, to increase immunogenicity of cancer vaccines and immune
responses in cancer immunotherapy, an injectable hydrogel based on PCLA-b-PEG-b-PCLA
copolymer-conjugated hyaluronic acid (HA) has been prepared and loaded with
immunomodulatory factors [32]. Cancer immunotherapy has been an attractive strategy for
treating cancers since it can restore self-specific immune responses to tumors and provide
tolerable and durable treatment regimens. As for conventional vaccines, there is a risk of
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undetected contamination from viruses or bacteria [33]. Thus, both inactivated and attenuated
vaccines need to be refrigerated during storage and transportation. DNA vaccines consisting of
plasmid DNA encoding disease-specific antigens have been proven to be safer than conventional
vaccines [34]. DNA vaccines have other advantages such as low cost, good thermal stability, and
ease of mass production. Despite the enormous potential of DNA vaccines, it is challenging to
stimulate sufficient immune responses by DNA vaccines themselves. Hence, efficient delivery
systems of DNA vaccines that can generate effective immune responses by actively modulating

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antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages need to be

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developed. Prolonging DNA vaccine retention time at target sites that contain abundant APCs

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can lead to higher possibilities of cell uptake of DNA vaccines and enhanced immune responses.
Recently, injectable hydrogels with crosslinked networks that undergo a sol-gel phase transition
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upon exposure to outer stimulating factors such as temperature have attracted a lot of attention
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for vaccine delivery. HA has been used as a popular material for constructing hydrogels with
desired properties. However, native HA is not serviceable due to its inclination of degradation in
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vivo and weak mechanical behaviors. One way to improve its durability and toughness is by
conjugating synthetic materials with HA. Our group has conjugated HA with
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temperature-responsive PCLA-b-PEG-b-PCLA copolymer and developed an injectable,

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spontaneously formed, and flexible hydrogel that contains microporous networks after
subcutaneous administration via a hypodermic syringe in a non-invasive manner [32]. After such
in situ-forming injection, the hydrogel reservoir is formed close to the dermis and plenty of local
APCs could migrate to the boundary of the hydrogel and infiltrate microporous networks.
Ovalbumin (OVA)-expressing DNA polyplexes inside the hydrogel could then be released and
taken up by these APCs due to continuous degradation of microporous hydrogels. Recruited
mature APCs could transfer to lymph nodes and generate corresponding immune effects.
Subsequent antibody dependent cell-mediated cytotoxicity (ADCC) can lead to effective
metastasis tumor suppression. This hybrid hydrogel has been highlighted as a potential DNA
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vaccine delivery platform that can induce powerful antigen-specific immune responses in vivo
for cancer immunotherapy.
Maturation and antigen presentation of DCs are highly related to subsequent activation of T
cells in immunotherapy. Owing to abundant DCs in subcutaneous layer and skin draining lymph
nodes, local immunomodulation provides more specific and more effective but less systemic
toxic treatment to promote immune responses than systemic immunomodulation. The
development of sustained release technology has greatly promoted the transformation of

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vaccines from traditional immunizations using multiple doses to immunizations using a single

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dose, thereby enabling vaccines to achieve highly efficient and durable immunity with lower

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toxicity through a single injection. Currently, injectable hydrogel is one of the most attractive
biomaterials because it is responsive to stimuli with tunable properties. Disadvantages of natural
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hydrogels include batch-to-batch variations and uncontrolled biodegradation, making it difficult
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to control their mechanical properties. In contrast, synthetic polymers generally have a

well-defined structure. They are highly reproducible. In addition, they can be conjugated with
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biomolecules. A polymer solution is a fluid at room temperature. It can be mixed with active
ingredients for administration by subcutaneous injection. In this regard, after evaluating a large
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number of polymers, we have designed an immune-stimulating hydrogel based on

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PCLA-b-PEG-b-PCLA-conjugated bovine serum albumin (BSA) (PCLA-b-PEG-b-PCLA/BSA

hydrogel) that is injectable. It can also be stably formed in situ (Fig. 1) [35]. Owing to the
enormous potential of DNA-based vaccines that can stimulate both humoral and cellular immune
responses, effective delivery of DNA-based vaccines to DCs has attracted a lot attention. In
addition, this hydrogel is suitable for delivering DNA-based vaccines owing to their good
biocompatibility, high loading capability, and gentle operating conditions. After subcutaneous
injection, it could attract and recruit DCs to micro-holes inside hydrogels without using any
immune adjuvant. Thus, DNA-based vaccines could be delivered to these DCs. Activated DCs
could then travel to skin draining lymph nodes. According to our results, amounts of recruited
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cells are significantly higher than those using traditional immune chemokine such as
granulocyte-macrophage colony-stimulating factor (GM-CSF). DNA vaccines encoded with
fusion proteins from microporous networks can produce strong antigen-specific immune
responses in vivo. Furthermore, the hydrogel could be gradually degraded under the skin. Loaded
DNA vaccine could be released and effectively taken up by DCs in the skin for a long term,
thereby triggering sufficient and specific immune responses.

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Figure 1. Schematic illustration of recruitment of host cells into PCLA-b-PEG-b-PCLA/BSA

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hydrogels and subsequent activation in response to DNA vaccine-bearing polyplexes released

from these hydrogels. Copyright 2019 from Elsevier [35].

In addition to vaccine delivery, PCLA-b-PEG-b-PCLA copolymer has been incorporated with

some other natural polymers to prepare in situ-forming and injectable hydrogels with good
temperature sensitivity and biocompatibility for many biomedical applications. For example, our
group has conjugated PCLA-b-PEG-b-PCLA copolymer with gelatin
(PCLA-b-PEG-b-PCLA/gelatin) and successfully prepared a temperature-responsive and
injectable hydrogel for wound healing [36]. By utilizing ECM mimicking property of gelatin and
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temperature-induced gelling ability of PCLA-b-PEG-b-PCLA, the

PCLA-b-PEG-b-PCLA/gelatin system can successfully seal excisional wounds without using
needing any growth factor. It is noteworthy that such PCLA-b-PEG-b-PCLA/gelatin conjugates
with a low content of PCLA-b-PEG-b-PCLA cannot form stable gels in vitro. However, higher
contents of PCLA-b-PEG-b-PCLA block copolymer in PCLA-b-PEG-b-PCLA/gelatin
conjugates are needed to create an ideal hydrophilic/hydrophobic balance to preserve the gel
morphology inside the body at 37°C. This indicates that the length of the PCLA-b-PEG-b-PCLA

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segment plays an important role in determining the hydrophilicity and hydrophobicity of

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conjugates. It has been shown that the injectable PCLA-b-PEG-b-PCLA/gelatin system has

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tremendous healing effect in both in vivo cutaneous wound model and excisional wound model.
Such PCLA-b-PEG-b-PCLA/gelatin hydrogel possesses adhesive and sealant properties on
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wounds, thus effectively healing wounds and finally achieving a smooth appearance that is
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similar to a normal skin.

In another PCLA-b-PEG-b-PCLA-based research, our group has engineered alginate with
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PCLA-b-PEG-b-PCLA to obtain a biocompatible injectable scaffold system for accelerating

bone biomineralization (Fig. 2) [37]. We have sequentially conjugated
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PCLA-b-PEG-b-PCLA-NH2 and O-phosphoryl ethanolamine via EDC/NHS chemistry to

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alginate (Alg) to create phosphorylated PCLA-b-PEG-b-PCLA/Alg bioconjugates. Such

PCLA-b-PEG-b-PCLA/Alg bioconjugates possess sol-to-gel transition when temperature is
changed from room condition to physiological condition. Their in situ biomineralization has been
investigated both in vitro and in vivo. In vitro tests were conducted using stimulated body fluids
for four weeks. XRD analysis confirmed that such PCLA-b-PEG-b-PCLA/Alg bioconjugate
hydrogel could induce the growth of crystalline hydroxyl apatite. Furthermore, in vivo tests
showed that such PCLA-b-PEG-b-PCLA/Alg bioconjugate hydrogel could sustainably release
mitogenic factors (BMP-2) for more than three days. BMP-2-containing
PCLA-b-PEG-b-PCLA/Alg bioconjugate hydrogel also showed much higher amounts of calcium
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deposition after four weeks in comparison with other experiment groups. These results suggest
that such BMP-2-containing PCLA-b-PEG-b-PCLA/Alg bioconjugate hydrogel has the ability to
escalate biomineralization under body conditions.

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Figure 2. Schematic illustration of ECM-mimicking injectable PCLA-b-PEG-b-PCLA/Alg

hydrogels encapsulated with BMP-2 and subsequent sustained release of BMP-2 for repair of
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bone defects. Copyright 2020 from Elsevier [37].

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3. Temperature and pH dual responsive injectable hydrogels

Although temperature-responsive and injectable hydrogels have shown great potentials in
different biomedical applications, these hydrogels still suffer from some problems during
preparation and injection. For example, it generally takes more than 24 hours for the
PCLA-b-PEG-b-PCLA copolymer to form a uniform sol even at low temperature, which makes
the preparation process time-consuming. Due to the high viscosity of the PCLA-b-PEG-b-PCLA
sol at room temperature, injecting PCLA-b-PEG-b-PCLA sol is sometimes difficult due to
possible needle clogging. To solve this problem, our group has developed a series of temperature
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and pH dual responsive injectable hydrogels [38-42]. These dual responsive polymers for the
preparation of injectable hydrogels contain many ionizable groups that make polymers easier to
be dissolved by adjusting pH. According to the different ionizable groups, dual responsive
polymers can be divided into cationic polymers and anionic polymers. Under an ionized state,
the ionic polymers have higher hydrophilicity, which makes it easier to inject their aqueous
solutions through needles. However, under a non-ionized state, the polymers exhibit amphiphilic
properties that induce gelation. Moreover, charges of dual responsive hydrogel networks provide

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these hydrogels additional functions due to interactions between the hydrogel matrix and loaded

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agents with net charges.

3.1 Cationic polymeric hydrogels -p

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Poly(ethylene glycol)-poly(β-amino ester urethane) (PEG-PAEU) multiblock copolymers are
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pH- and temperature-responsive biodegradable copolymers that can undergo sol-to-gel

transitions at body conditions [43, 44]. PEG segments can enhance biological half-lives and
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hydrophilicity. They can also improve pharmacokinetic properties and immunogenicity of

PEG-PAEU copolymers [45, 46]. PAEU blocks endow pH and temperature sensitivity to
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PEG-PAEU copolymers. Under acidic pH and room temperature, both PEG blocks and ionized
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PAEU blocks are hydrophilic, making PEG-PAEU copolymers fully soluble in water. On the
other hand, under body conditions (pH 7.4 and 37oC), tertiary amine groups in PAEU blocks are
deionized, causing hydrophobic interactions and hydrogen bonding between deionized PAEU
blocks that can then induce physical crosslinking and turn the copolymer solution to a semi-solid
hydrogel [47]. Due to their high water contents, biodegradability, biocompatibility, and in situ
gel forming ability, PEG-PAEU copolymers are considered as good candidates for delivering
therapeutic agents with other medical applications [48, 49]. In recent years, we have developed
diverse PEG-PAEU-based hydrogels for biomedical applications.
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In a report in 2017, we have coupled PEG-PAEU to sulfhydryl groups of human serum

albumin (HSA) [50]. Hydrogels based on as-synthesized PEG-PAEU/HSA hybrid copolymer
can greatly improve biostability and circulation half-life of biological drugs (Fig. 3) [50].
Site-specific conjugation of pH and temperature dual responsive PEG-PAEU to human-sourced
HSA could be utilized for controlled delivery of therapeutic proteins. Multiple hydrophobic
cores of HSA could entertain hydrophobic molecules through physical binding without altering
their bioactivities. The PEG-PAEU/HSA hybrid hydrogel system has been used to deliver

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uricase enzyme (Uox) for treating hyperuricemia-associated diseases. Moreover, Uox can be

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conjugated with palmitic acid (PA) to make Uox-PA complexes. This favors specific binding of

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enzymes within protein shells and shielding in PEG-PAEU/HSA hybrid hydrogel systems.
Compared to native Uox, Uox-loaded hybrid hydrogels can maintain the bioactivity of Uox and
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extend pharmacokinetics profile. It is noteworthy that Uox-loaded hybrid hydrogels can escalate
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the pharmacological efficacy in hyperuricemia mice. It has been found that hybrid hydrogels
administered formulation can rapidly reduce the level of uric acid and normalized the level of
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uric acid within 5 hours after administration. After 12 hours, serum uric acid levels in hybrid
hydrogel-administered mice were similar to those in normal mice without hyperuricemia.
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Overall, these results indicate that the combined system of Uox-PA and hybrid hydrogels can
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effectively normalize levels of uric acid in hyperuricemic mice. In another attempt to extend the
activity and half-life of Uox, we have conjugated albumin-binding peptide (ABP) (femtomolar
dissociation constant), an engineered albumin-binding domain originally derived from protein G
of Streptococcus species, to PEG-PAEU to create temperature and pH dual responsive
PEG-PAEU/ABP bioconjugate (Fig. 4) [51]. Uox was conjugated with HSA to generate
HSA-conjugated Uox (Uox-HSA). It was found that the Uox-HSA-encapsulated
PEG-PAEU/ABP hydrogels could prolong serum activity for more than 144 hours. It was
noteworthy that the serum activity and half-lives of Uox-HSA encapsulated in PEG-PAEU/ABP
hydrogels were over 80-fold higher than those of the control group (Uox). In vivo serum activity
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and half-lives of Uox-HSA encapsulated in PEG-PAEU/ABP hybrid hydrogels also showed a

uric acid lowering effect for a prolonged period of time (over 72 hours) in
hyperuricemia-induced mice. These results suggest that the combination of HSA and ABP
introduced into therapeutic proteins and PEG-PAEU hydrogel systems can sustainably deliver
therapeutic proteins and reserve therapeutic effects for a long period of time.

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Figure 3. Schematic diagram showing Uox-loaded PEG-PAEU/HSA hydrogels and their

controlled release of Uox for degradation of uric acid crystals in hyperuricemia mice models.
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Copyright 2017 from Elsevier [50].

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Figure 4. Schematic diagram showing sustained release of Uox-HSA from PEG-PAEU/ABP
hybrid hydrogels and prolonged serum half-life for hyperuricemia treatments. Copyright 2020
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from Elsevier [51].

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In another report in 2017, PEG-PAEU hydrogels were employed for cancer cell-specific
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delivery of cisplatin (CDDP)-bearing chondroitin sulfate (CS) nanogels (CS-Nanogels) [52].

CDDP is an anticancer agent that is frequently employed for treating many kinds of solid tumors
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in cancer chemotherapy. To raise safety profiles and overcome multidrug resistance, we

introduced CS-nanogels prepared by crosslinking reactions between CS-amino malonic acid
conjugates and CDDP by chelating ligand-metal coordination. The hydrogel network contained
CS-nanogels with strong ionic interactions between tertiary amine groups of PEG-PAEU and
CS-nanogels. Such CS-nanogels-encapsulated hydrogel complex exhibited more sustainable
release of CDDP than CDDP-encapsulated hydrogel due to the presence of a double-layered
depot system by both metal-ligand coordinated bonds and ionic interactions. Both
CD44-negative (NIH3T3) cells and CD44-positive (A549) cells were used to study
CD44-mediated cell uptake of CS-nanogels. Experiment results showed that CS-nanogels
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enhanced cellular uptake efficacy and targeted into CD44-positive A549 cells. On the other
hand, in vitro cancer cell-specific cytotoxicity results showed that CS-nanogels had slower
anticancer activity than free CDDP. This indicated that CS-nanogels exhibited slower
internalization into cells and slower release process of CDDP from nanogels after cell uptake via
endocytosis than free CDDP. After incubation for 48 hours, experiment results showed that
CS-nanogels could improve cell growth inhibition of lung cancers, indicating a sustainable
release of CDDP over time. In vivo gel formation experiments confirmed that

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CS-nanogels-loaded hydrogel precursors could form a soft gel in situ and that as-formed gels

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were bioresorbable within 8 weeks after subcutaneous injection without causing any
inflammation.
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In another application, PEG-PAEU copolymer was conjugated with HA to synthesize a hybrid
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polymer, PEG-PAEU/HA [53]. Hydrogel prepared from PEG-PAEU/HA hybrid copolymer was
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used as a biomimic injectable bio-hydrogel (IBHG) system that could spontaneously form a gel
at inflammatory or wound sites with adjustable hydrogel surface control to meet different
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requirements of wound healing. Interestingly, such PEG-PAEU/HA IBHG system could inhibit
burst release, prolong the release of human growth hormone (hGH) in a sustained manner, and
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subsequently increase the bioavailability of hGH. It was found that such hydrogel formulations
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could prolong the therapeutic level of hGH for more than a week. Open wounds treated with
IBHGs showed better wound closing results than PEG-PAEU hydrogels. This can be explained
by the fact that HA is a naturally occurring non-immunogenic natural polysaccharide.
Furthermore, skin tissue sections of the IBHG-treated group with H&E staining showed
complete re-epithelialization and more epithelial cells. In addition, the presence of high-density
collagen fibers was confirmed by Masson’s trichrome staining of skin tissue sections. These
improved wound healing properties confirmed that IBHG could be utilized as a bioresorbable,
biocompatible, and in situ fillable wound healing formulation for excisional wounds.
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Stimuli-sensitive polypeptides are emerging artificial protein polymers. They have attracted
increased attention because of their promising applications in drug delivery and cancer diagnosis
considering their biodegradability, biocompatibility, and bioactivity [54, 55]. In practice,
molecular weights and chemical structures of polypeptides can be adjusted by different synthesis
methods, resulting in more functional properties for different biomedical purposes [56, 57].
Moreover, they can undergo reversible secondary conformation transformations and/or
hydrophilic/hydrophobic transformations under endogenous conditions such as reduction, pH,

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and enzymes or exogenous stimuli such as temperature and light [58]. Therefore, stimuli‐

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sensitive synthetic polypeptides have been widely applied to construct various drug carriers

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including nanogels, micelles, and hydrogels for drug delivery applications [59]. For example, our
group has synthesized novel polyglutamate derivatives with excellent pH and temperature
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sensitivities for in vivo delivery of proteins (Fig. 5) [60]. A triblock polyglutamate-based
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copolymer, poly [(2-(dibutylamino)

ethyl-L-glutamate)-co-(γ-benzyl-L-glutamate)]-b-PEG-b-poly [(2-(dibutylamino)
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ethyl-L-glutamate)-co-(γ-benzyl-L-glutamate)] (PNLG-co-PBLG-b-PEG-b-PBLG-co-PNLG),
was synthesized by a ring-opening polymerization followed by aminolysis reaction. Aqueous
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solution of PNLG-co-PBLG-b-PEG-b-PBLG-co-PNLG appeared as low viscoelastic sol at low

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pH and room temperature while its hydrophobic PBLG and cationic PNLG blocks would
crosslink together and transfer the sol into a hydrogel under physiological conditions (pH 7.4 and
37°C) due to a change in hydrophilic/hydrophobic balance. Subcutaneous injection of such
polymer sol into backs of Sprague-Dawley (SD) rats has led to in situ gel formation and
bioabsorbability of copolymers. Moreover, these polypeptide-based triblock copolymers could
easily form a viscous gel when it is loaded with human growth hormone (hGH), thus inhibiting
the initial burst release of hGH and prolonging the release time of hGH. Therefore, they have
superior bioresorbable and sustained protein release features. They are good candidates for
therapeutic protein delivery.
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Figure 5. Schematic diagram showing injectable and hGH-loaded

PNLG-co-PBLG-b-PEG-b-PBLG-co-PNLG hydrogels and their application for protein delivery.
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Copy right 2017 from Royal Society of Chemistry [60].

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3.2 Anionic polymeric hydrogels

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In addition to cationic copolymers containing tertiary amine groups, temperature and pH dual
responsive injectable hydrogels can also be prepared from anionic copolymers containing
sulfonamide groups [61-63]. Different from cationic copolymers, anionic copolymers undergo
ionization to de-ionization transition upon decreasing polymer solution pH from high pH to
physiological pH. This transition induces a change of anionic copolymers from hydrophilic to
amphiphilic state, causing gelling of a concentrated polymer solution. Compared with cationic
copolymers-based injectable hydrogels, anionic polymers-based injectable hydrogels are
favorable for loading agents that are insoluble or unstable at low pH. A new type of in
situ-forming bioresorbable injectable hydrogel bearing anionic sulfonamide groups has been
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reported for sustained delivery of proteins in vivo [64]. First, a series of triblock polyglutamate
derivatives were synthesized by ring-opening polymerization and side chain modification. These
sulfonamide groups-containing block copolymers are soluble at room temperature and high pH
while they are able to form hydrogels at body conditions (37°C and pH 7.4). By controlling
chemical compositions of these polypeptides, physical properties of hydrogels made from these
polypeptides can be precisely tuned. Anionic properties make hydrogels suitable for loading
cationic proteins (lysozyme as a model) through electrostatic interaction and subsequently

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continuous release. Lysozyme-encapsulated polyglutamate derivatives-containing sols could

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form viscoelastic gels in vivo easily and release lysozyme slowly for about a week. Subsequent

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cytotoxicity studies have confirmed the safety of utilizing these hydrogels for biomedical
applications. Subcutaneous injection of polyglutamate derivative sols into backs of SD rats at
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room temperature resulted in the formation of stable and round-shape gels. These in situ-forming
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gels were bioresorbable in six weeks without causing inflammation at the injection site. These
results indicate that using anionic polymeric hydrogels provides a convenient and effective
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approach to deliver positively charged proteins for future protein therapeutics.

By connecting sulfonamide groups-containing oligomers to both ends of triblock copolymer
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PCL-b-PEG-b-PCL or PLA-b-PEG-b-PLA, pentablock copolymers with both pH and

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temperature sensitivities could be synthesized [65]. Gelling properties of these pentablock

copolymers could be well-controlled by tuning lengths of sulfonamide groups-containing
oligomers. Upon subcutaneous injection, soft gels could be formed in situ. These gels are then
degraded slowly for several weeks. These injectable hydrogels can serve as excellent depot for
chitosan-insulin electro-sprayed nanospheres without changing bio-properties of insulin. Upon
degradation of these hydrogels and sustained release of insulin, blood concentration of insulin
could be maintained at a stable level and blood glucose level could be effectively reduced. These
chitosan-insulin electro-sprayed nanospheres-loaded injectable hydrogels are good candidates for
treating type 1 diabetes [66].
 Journal Pre-proof

To increase contents of sulfonamide groups in anionic copolymers and improve mechanical

properties of injectable hydrogels, a series of sulfamethazine-containing multiblock
polyurethanes (PUSMA) were synthesized by step polymerization using hexamethylene
diisocyanate as a linker. Networks of injectable hydrogels prepared from these polymers are
negatively charged because not all sulfonamide groups are de-ionized. Thus, these hydrogels are
suitable for sustained release of cationic proteins. They can avoid initial burst release due to
electrostatic interactions between the anionic hydrogel network and cationic proteins [67]. By

of
encapsulating DNA-loaded cationic polyplex, an anionic PUSMA copolymer can be employed to

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accelerate wound healing (Fig. 6) [68]. It has been found that PUSMA copolymer has good

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tissue adhesive properties possibly due to the presence of urethane ester groups in polymer
backbones. Outstanding adhesive properties of PUSMA injectable hydrogels allowed these
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hydrogels to closely adhere to the wound and provide an aqueous matrix to accelerate wound
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healing. Compared to PUSMA hydrogels without loading of DNA-containing polyplex,

DNA-loaded cationic polyplex-encapsulated PUSMA hydrogels showed better wound healing
na

results. To further improve the sustained release ability of anionic PUSMA hydrogels, heparin
was incorporated with PUSMA copolymer for affinity-based sustained release of bioactive
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factors [69]. Owing to the affinity between heparin and vascular endothelial growth factor
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(VEGF), heparin-incorporated PUSMA injectable hydrogels showed much longer sustained

release of VEGF than pure PUSMA hydrogels. Such prolonged release of VEGF induced
generation of more blood vessels throughout the whole hydrogel network.
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Figure 6. Schematic illustration showing dual responsive and injectable PUSMA hydrogels and
their applications in wound healing. Copyright 2018 from American Chemical Society [68].
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Although PUSMA injectable hydrogels have anionic matrices, they can also be used for local
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delivery of anionic therapeutic agents such as oncolytic adenoviruses (Ads) (Fig. 7) [70]. It has
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been found that PUSMA injectable hydrogels could provide a friendly environment for Ads and
preserve their bioactivity for almost two weeks. In comparison with naked Ads, Ads loaded
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within injectable PUSMA hydrogels exhibited stronger cytotoxicity to cancer cells even after
eleven days. In human xenograft tumor models, such Ad-loaded PUSMA injectable hydrogels
also showed improved and long-acting tumor growth inhibition. These outstanding properties
make injectable and Ad-loaded PUSMA hydrogels very promising for effective and long-lasting
cancer treatment.
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Figure 7. Schematic illustration showing PUSMA hydrogels and their applications in sustained
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release of oncolytic Ads for cancer therapy. Copy right 2019 from Royal Society of Chemistry
[70].
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4. Summary and Outlook

In summary, inspired by Prof. Sung Wan Kim’s pioneering work in the field of biodegradable
and injectable hydrogels, we have developed a series of advanced temperature-responsive and
temperature/pH dual responsive hydrogels for different biomedical applications. Table 1
summarizes several important thermo-sensitive and dual sensitive injectable hydrogels
introduced in this review. By incorporating natural polymers with PCLA-b-PEG-b-PCLA,
properties of PCLA-b-PEG-b-PCLA injectable hydrogels are improved. Their applications have
been extended. Furthermore, by introducing tertiary amine groups or sulfonamide groups into
polyurethane or polypeptides, temperature and pH dual responsive hydrogels could be prepared.
 Journal Pre-proof

The presence of ionizable groups can successfully lower the viscosity of polymer sols, making it
easier to inject them into deep tissues. Charges in the hydrogel matrix make these hydrogels
advantageous for loading, attracting, and releasing charged agents.

No. Type Polymer Application Ref.

Natural
PCLA-b-PEG-b-PCLA/

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1 polymers-incorporated DNA vaccine delivery 35
BSA
hydrogel

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Natural
PCLA-b-PEG-b-PCLA/
2 polymers-incorporated Bone regeneration 37
hydrogel
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Alg
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Natural
Hyperuricemia
3 polymers-incorporated PEG-PAEU/HSA 50
treatment
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hydrogel

Natural
Hyperuricemia
4 polymers-incorporated PEG-PAEU/ABP 51
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treatment
hydrogel
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PNLG-co-PBLG-b-PEG
5 Dual responsive hydrogel Protein delivery 60
-b-PBLG-co-PNLG
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Wound healing and

6 Dual responsive hydrogel PUSMA 68, 70
cancer therapy

Table 1. Summary of several important thermo-sensitive and dual sensitive injectable hydrogels.

Although injectable hydrogels have shown great potentials in several different biomedical
applications, problems still remain, thus hindering applications of injectable hydrogels. First,
solid contents of most physically crosslinked injectable hydrogels are generally high (10 wt% -
30 wt%), which increases the cost of using injectable hydrogels. It might also cause some
 Journal Pre-proof

discomfort to patients. Thus, developing injectable hydrogels with low solid content is necessary.
Loh’s group and Jeong’s group have reported thermosensitive hydrogels with low solid contents
based on poly(ethylene-butylene) and polyalanine/phenylalanine (PAF), respectively [71, 72].
The solid contents of their hydrogels can be as low as 2-3 wt%, however, solid contents of other
well-known thermosensitive hydrogels are still very high. Second, polymer sols with lower
viscosity before injection and faster gelling speed need to be developed. Lower viscosity of
polymer sols can avoid needle clogging and faster gelling speed can avoid polymer sol from

of
moving to undesired sites. Third, the release of loaded agents highly depends on the degradation

ro
rate of hydrogels. Thus, more studies about degradation control of injectable hydrogels need to

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be conducted. Fourth, more stimuli that could trigger the gelling of polymer sols should be
explored so that injectable hydrogels can be used in different situations. Fifth, storage is a very
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important issue for polymer sols. It should be considered for real products, especially the
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degradation problem during storage. Although thermogelling PEG/PAF aqueous solutions did
not show degradation at room temperature for one month, degradation problem during storage
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still needs to be improved for most polyester-based thermogels. Sixth, more functional groups
should be incorporated into injectable hydrogels to extend application areas of hydrogels. With
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fast development of injectable hydrogels, we believe that patients will have more and more
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powerful and economic products in the near future.

Acknowledgement
This research was supported by National Natural Science Foundation of China (NSFC) (No.
81901900; 21805108) and Basic Science Research Program through a National Research
Foundation of Korea grant funded by the Korean Government (MEST)
(NRF-2017R1A5A1070259).

Credit Author Statement

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Yi Li: Writing - Original draft

Hong Yu Yang: Writing - Review & editing

Doo Sung Lee: Funding acquisition; Supervision

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