Effect of hydrophobic polypeptide length on performances of thermo-sensitive hydrogels

Article abstract of DOI:10.3390/molecules23051017

Thermosensitive gels are commonly used as drug carriers in medical fields, mainly due to their convenient processing and easy functionalization. However, their overall performance has been severely affected by their unsatisfying biocompatibility and biode

Full text of DOI:10.3390/molecules23051017

molecules
Article
Effect of Hydrophobic Polypeptide Length on
Performances of Thermo-Sensitive Hydrogels
Jiandong Han 1,2 ID , Xingyu Zhao 2, Weiguo Xu 2, Wei Wang 1,*, Yuping Han 2,3,* and
Xiangru Feng 2,
*
ID
1
Department of Chemistry, Changchun University of Science and Technology, Changchun 130022, China;
jdhan@ciac.ac.cn
Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry,
Chinese Academy of Sciences, Changchun 130022, China; star20012002@163.com (X.Z.);
wgxu@ciac.ac.cn (W.X.)
2
3
Department of Urology, China-Japan Union Hospital of Jilin University, Changchun 130033, China
Correspondence: weiwanglg@163.com (W.W.); hyp181818@126.com (Y.H.); xrfeng@ciac.ac.cn (X.F.)
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 12 April 2018; Accepted: 21 April 2018; Published: 26 April 2018
Abstract: Thermosensitive gels are commonly used as drug carriers in medical fields, mainly due
to their convenient processing and easy functionalization. However, their overall performance has
been severely affected by their unsatisfying biocompatibility and biodegradability. To this end,
we synthesized poly(L-alanine) (PLAla)-based thermosensitive hydrogels with different degrees
of polymerization by ring-opening polymerization. The obtained mPEG45 PLAla copolymers
−
showed distinct transition temperatures and degradation abilities. It was found that slight changes
in the length of hydrophobic side groups had a decisive effect on the gelation behavior of the
polypeptide hydrogel. Longer hydrophobic ends led to a lower gelation temperature of gel at the
same concentration, which implied better gelation capability. The hydrogels showed rapid gelling,
enhanced biocompatibility, and better degradability. Therefore, this thermosensitive hydrogel is a
promising material for biomedical application.
Keywords: amphiphilicity; phase change; polyamino acids; degradability
1. Introduction
The application of hydrogels in the biomedical field has rapidly increased over the past
decade, including use in three-dimensional (3D) cell culture [
1–3], drug delivery [4–6], and tissue
engineering [ ]. Polymer hydrogels can trap a large amount of moisture inside for easier cell
7–9
membrane penetration and drug transmission. They enjoy excellent physical properties and exhibit a
controllable degradation process. On account of the incomparable convenience, the in situ gelation
of biodegradable hydrogels has aroused great interest in many researchers. Among numerous
biodegradable hydrogels, thermosensitive hydrogels have specific advantages. On the one hand,
they are rather safe for clinical utilization because the common thermosensitive gel preparation process
does not involve the use of organic solvents. On the other hand, the gelling conditions of heat-sensitive
hydrogels are easy to control, making them applicable in the field of biomedicine [10–12].
Thermosensitive hydrogels can be generated from block copolymers consisting of hydrophilic
poly(ethylene glycol) (PEG) and hydrophobic moieties such as poly(lactic acid) (PLA) [13
poly(lactic-co-glycolic acid) (PLGA) [15 17], poly( -caprolactone) (PCL) [18 20], polyamidoamine
(PAMAM) [21], and so on. Thermosensitive hydrogels are soluble at low temperatures, which facilitates
the introduction of chemotherapeutic drugs [22 24], functional proteins [25], and cells. Due to
the amorphous form of the hydrogel at low temperatures, it can be seamlessly filled into injury
,14],
–
ε
–
–
Molecules 2018, 23, 1017; doi:10.3390/molecules23051017
www.mdpi.com/journal/molecules

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sites [26,27]. At body temperature, the soluble hydrogels turn into a gel state and provide a depot for
the encapsulated medical agents to achieve sustained release and long-term therapeutic effect [28].
For example, growth factors can be slowly and continuously released locally for the efficient repair
of bone and nerve tissues [29–31]. The hydrogels can also function as 3D scaffolds and as a nutrient
resource for cell growth in tissue engineering [32], aiming to help cells grow in a more uniform manner
and promote cell proliferation [33].
In 2012, Qian’s team prepared a bionic hydrogel composed of three components (i.e., triblock
PEG
satisfactory efficacy in skull repair [34]. In 2015, Chen’s team engineered a thermosensitive
PLGA PEG PLGA hydrogel loaded with 5-fluorouracil for the prevention of postoperative tendon
−
PCL
−
PEG copolymer (PECE), nano-hydroxyapatite (n-HA), and collagen), which showed
−
−
adhesion, and acquired a good histological score [35]. However, despite the wide application of
thermosensitive hydrogels in various diseases in clinical practice, they are not free from flaws.
Some hydrogels are difficult to gelatinize, and the degradation time is uncontrollable. Moreover,
hydrogels with poor biocompatibility always lead to severe tissue damages. Therefore, there is
an urgent need to develop thermosensitive polypeptide polymers with good biocompatibility and
biodegradability to avoid the above problems.
Towards this aim, peptide systems have been developed to avoid evoking the formation
of an acidic microenvironment during degradation, and thus reduce the damages to
surrounding tissues and minimize the adverse effects toward the bioactivity of the loaded
protein or cells. Apart from this, the gelation performance of the polypeptide copolymer
can be tuned by copolymerization with various hydrophobic or hydrophilic amino acid
monomers, which widens the clinical applications. In 2011, Jeong’s team reported the synthesis
of poly(alanine-co-leucine)
−
poloxamer
−
poly(alanine-co-leucine) (PAL
−
PLX PAL) hydrogels,
−
which exhibited a sustained drug release pattern without producing evident inflammatory effects [22].
In 2017, methoxy poly(ethylene glycol)-block-poly(L-alanine-co-L-phenylalanine) mPEG-b-PLAF
hydrogel was synthesized by the ring-opening polymerization (ROP) reaction of L-alanine
N-carboxyanhydrides (L-Ala NCA) and L-phenylalanine NCA, with amino-terminated mPEG
(mPEG-NH2) as a macroinitiator. Encapsulated with combretastatin A4 (CA4) and doxorubicin
(DOX), mPEG
melanoma cells [36].
−
b PLAF hydrogel had an excellent inhibitory effect on the proliferation of B16F10
−
In this work, mPEG
−
poly(L-alanine) (mPEG
−
PLAla) polymers with different degrees
PLAla30, mPEG45 PLAla22,
PLAla14. The influence of different DPs on the gelation properties of hydrogels was
of polymerization (DPs) were synthesized and named mPEG45
and mPEG45
−
−
−
studied. It was found that hydrogels with higher DP showed better gelation ability than those with
lower DP. In addition, the gelation behavior, tissue safety, and cytotoxicity of these polymers in vitro
and in vivo were also tested and evaluated. The study revealed that these hydrogels had notable
potential for employment in the biomedical field.
2. Results and Discussion
2.1. Material Synthesis and Structure Characterization
Triphosgene and L-alanine were synthesized into L-Ala NCA in dried tetrahydrofuran (THF)
solvent, as shown in Scheme 1. The polypeptide hydrogels were produced via the ROP of L-Ala NCA
initiated by mPEG45-NH2. The mPEG45
by altering the feed amount. The DPs of PLAla were determined by contrasting the integral of the
methyl peaks of side chains ( CH3) with the methylene peak of PEG ( CH2CH2O ). The DPs of
mPEG45−PLAla30, mPEG45−PLAla22, and mPEG45−PLAla14 were 30, 22, and 14, respectively.
−
PLAla copolymers with different DPs were synthesized
−
−
−

Molecules 2018, 23, 1017
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Scheme 1. Synthetic routes of L-alanine N-carboxyanhydrides (l-Ala NCA) and methoxy poly(ethylene
glycol)−poly(l-alanine) (mPEG45−PLAla).
The typical proton nuclear magnetic resonance (1H-NMR) spectrum of block copolymer is shown
in Figure 1a, and all peaks could be accurately assigned. The peaks at 1.47, 3.40, 3.50–3.80, and 4.64 ppm
stood for the protons of alanine methyl, mPEG terminal methoxy, mPEG backbone, and polymer
methine, respectively, indicating the successful synthesis of the three polymers.
(a)
(b)
Figure 1. Structure characterization of mPEG45
−
PLAla. (
a
) 1H-NMR spectra of mPEG45
PLAla30,
−
mPEG45
(DP); (
and mPEG45−PLAla14
−
PLAla22, and mPEG45
) Fourier-transform infrared (FT-IR) spectra of mPEG45
−
PLAla14, where the subscripts indicate the degree of polymerization
PLAla30, mPEG45 PLAla22,
b
−
−
.
The secondary structure o−f1the copolymer was studied by FT-IR. In Figure 1b, characteristic
−1
peaks of amide bond at 1627 cm and 1544 cm were observed, indicating that all copolymers went
through the main β-sheet conformation.
2.2. Gelation Ability and Internal Appearance
The tube inversion method was used to determine the transition temperatures. Polypeptides
were solubilized in a phosphate-buffered saline (PBS) solution, and went through a solution–gel
transition with the change in temperature. The samples were defined as gel if they stayed still
when the vial was inverted for as long as 30 s. The phase diagrams of mPEG45
mPEG45 PLAla22 copolymers are shown in Figure 2a,b. Only copolymers with a DP of no less than
22 could undergo solution–gel transition at the concentration of 3.0–8.0 wt. %. It was noticeable that
−
PLAla30 and
−
mPEG45
−
PLAla22 showed a higher gel transition temperature than mPEG45
PLAla30 and mPEG45
−
PLAla30. The criti◦cal
◦
gelation temperatures (CGTs) of mPEG45
respectively, at the concentration of 5.0 wt. %. The lower CGT of mPEG45 PLAla30 could be explained
−
−
PLAla22 were 5 C and 15 C,
−
by longer hydrophobic end than that of mPEG45−PLAla22 [37].
Solution phase behaviors of the three copolymers at different temperatures were investigated.
The images in Figure 2a,b showed the mPEG45
−
PLAla30 and mPEG45
−
PLAla22 solutions in
PBS (5.0 wt. %) at 4 ◦C and 37 ◦C. Although all copolymers were dispersed in PBS at 4 ◦C,
the mPEG45
−
PLAla14 formed a clear solution while mPEG45
−
PLAla30 and mPEG45
−
PLAla22 showed
PLAla22 were
◦
a turbid state. When temperature rose to 37 C, mPEG45
−
PLAla30 and mPEG45
−

Molecules 2018, 23, 1017
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observed to be stably gelled within 5 min, while mPEG45
−
PLala14 remained in a viscous flow state.
This was because mPEG45
mPEG45 PLAla30 hydrogel (5.0 wt. %) was observed by scanning electron microscope (SEM). As can
be seen in Figure 2c, it was found that there were numerous tiny pores in the mPEG45 PLAla30 gel,
which was due to the 3D network structure of the gel. These pores showed a size of about 100 m and
were evenly distributed within the hydrogel, making them suitable as a depot for therapeutic agents.
These findings prove that mPEG45 PLAla30 and mPEG45 PLAla22 hydrogels feature good stability
−
PLala14 had better water solubility. Further, the morphology of the
−
−
µ
−
−
at body temperature and are suitable for biomedical applications.
(a)
(b)
(c)
(e)
(d)
(f)
) Solution–gel phase diagrams of the
Figure 2. Gelatinization characteristics of mPEG45
−
PLAla. (
a
mPEG45
(SEM) image of mPEG45
mPEG45 PLAla22 (PLAla22), and mPEG45
in phosphate-buffered saline (PBS) solution; (
−
PLAla30 and (
b
) mPEG45
−
PLAla22 copolymer solutions; (
c) Scanning electron microscope
◦
−
PLAla30 hydrogels formed at 40 C; (
d
) G’ of the mPEG45
PLAla14 (PLAla14) at the concentration of 5.0 wt. %
) Changes of G’ and G” of mPEG45 PLAla30 and
−
PLAla30 (PLAla30),
−
−
e
−
(f) mPEG45−PLAla22 in PBS solutions (5 wt. %).
2.3. Mechanical Performance Test
0
Thermally induced storage modulus (G ) and loss modulus (G”) changes of the three copolymers
were obtained by dynamic mechanical analysis. G0 represents the systematic gel-like behavior of
the elastic component of the complex modulus, and G” is an index of the viscous component of
the complex modulus and a measure of the sol-like behavior. The intersection of G0 and G” reflects
the sol–gel transition. As shown in Figure 2d, the G0 of mPEG45
obviously increased along with rising temperatures. However, only a minor increment in G0 was
−
PLAla30 and mPEG45
−
PLAla22

Molecules 2018, 23, 1017
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seen in mPEG45
−
PLAla14, and no significant change in G0 was detected, even when the temperature
◦
rose to about 50 C, implying that there was no sol–gel modification of mPEG45 PLAla14 within
the experimental temperature range. The reason was that hydrophilic mPEG was longer than
the hydrophobic end made up of polypeptides. Therefore, with a longer hydrophilic segment,
−
mPEG45
−
PLAla14 showed better water solubility than mPEG45
−
PLAla30 and mPEG45 PLAla22,
−
thus featuring less obvious sol–gel change. Notably, the length of the hydrophobic side chain had a
remarkable effect on the gelation function of the mPEG−peptide block copolymer.
◦
In Figure 2e,f, G0 was smaller than G” when the temperature was lower than CGTs (5 C in
mPEG45
−
PLAla30 and 12 ◦C in mPEG45
PLAla22), which reflected the viscous state of hydrogels.
−
As the temperature increased above CGTs, G0 was sharply elevated and surpassed G”, confirming the
gel formation. This result was in accordance with the findings in Figure 2a,b.
2.4. Mechanism of Gelatinization
In order to study the mechanism of sol–gel transition, the nuclear magnetic peak changes, diameter
changes, and conformation evolution of polypeptides in response to temperature were tested by carbon
nuclear magnetic resonance (13C-NMR), dynamic light scattering◦(DLS), and circular dichroism (CD).
As shown in Figure 3a, when temperature rose from 20 to 60 C, the characteristic peak of PEG
gradually moved from 69.7 to 70.3 ppm, which revealed continuous dehydration of the PEG block
during the heat-induced sol–gel transition process. Two main reasons accounted for the gelation of
polymers. First, the interaction between hydrophobic blocks became stronger as temperature increased.
Secondly, dehydration of PEG segments facilitated the aggregation of the hydrogels [38].
(a)
(b)
(c)
Figure 3. Gelation mechanism of the hydrogels.
(
a)
13C-NMR spectra (in D2O) of 5.0 wt. %
mPEG45 PLAla30 solution as a function of temperature; (
−
b
) Average hydrodynamic diameter (Dh)
g mL−1); (
) Circular
PLAla30 (0.05 mg mL−1) in aqueous solution as a function
of micelles of mPEG45 PLAla30 as a function of temperature in water (5.0
−
µ
c
dichroism (CD) spectra of mPEG45
of temperature.
−
As seen in Figure 3b, the diameter changes of mPEG45
−
PLAla30 were determined at the
PLAla30
◦
concentration of 5.0
µ
g mL−1. At 10 C, the average hydrodynamic diameter (Dh) of mPEG45
−
was about 31.4 nm. When the temperature rose to 20 ◦C and 50 ◦C, the number swiftly increased to
95.9 nm and 356.2 nm, respectively. The dramatic changes in particle size could be explained by the

Molecules 2018, 23, 1017
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interaction between the shell of PEG and core of polypeptides caused by the dehydration processof
PEG [38].
In Figure 3c, the CD spectrum illustrated the alteration of the secondary structure of the aqueous
mPEG45
−
PLAla30 during the sol–gel transition process. The two typical bands corresponding to
the -sheet conformation, a positive Cotton band at 195 nm and a negative Cotton band at 226 nm,
β
were clearly shown. The above results revealed that the synthesized thermosensitive hydrogel had
good physicochemical properties and a clear solution−gel transformation mechanism.
2.5. Degradability Test and Pathological Analysis
Since hydrogels are often used in the biomedical field, biodegradability is a decisive factor for
clinical application. If the degradation rate of the hydrogel is too high, it will lead to a rapid release
of the payload. In contrast, if the hydrogel requires a long time to degrade, then it will probably
remain at the injection site even after all drugs are released, which is inconvenient for further treatment.
The in vitro degradation of mPEG45
with elastase K or -chymotrypsin. As shown in Figure 4a, there was a mass loss of over 70% and
67% hydrogel in the elastase K and -chymotrypsin groups on day 15, respectively—considerably
higher than 20% in PBS. The results could be explained by the absence of elastase K or -chymotrypsin,
where the mass loss of gels was merely attributed to the surface erosion of the hydrogels. However,
with elastase K or -chymotrypsin, the polypeptide chains of the hydrogels were also fast degrading,
which sped up the loss of gels.
−
PLAla30 hydrogel (5.0 wt. %) was evaluated in PBS and PBS
α
α
α
α
(a)
(b)
(c)
Figure 4. Degradation of hydrogel in vitro and in vivo, and histological analysis of skin. (
a) Mass
loss curves−o1f in vitro degradation of hydrogels in PBS, and PBS with elastase-K or
α-chymotrypsin
(0.2 mg mL
) groups; (b) Images of in vivo gel maintenance at 10 min, 7, 14, 21, and 28 days after the
injection of 5.0 wt. % mPEG45 PLAla30 hydrogels; (c) Hematoxylin and eosin (H&E) images of the
−
skin tissue near the hydrogels on day 7, 14, 21, and 28, respectively.
The degradation process of hydrogels in vivo was monitored for 28 days, and images were taken
◦
at 10 min, 7, 14, 21, and 28 days after hydrogel treatment. At 4 C, 500.0
µ
L of mPEG45 PLAla30
−
solution (5.0 wt. %) was subcutaneously injected into Sprague Dawley (SD) rats through 21-gauge
−

Molecules 2018, 23, 1017
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syringe needles. In Figure 4b, the solution rapidly turned into gel in 10 min after injection. Seven days
later, only a small portion of the hydrogel was degraded. On day 14, the size of the hydrogel shrank
to less than 50%, while on day 28, the hydrogel was completely degraded. This degradation rate of
hydrogel was suitable for application in biomedical fields requiring long-term treatment, such as tissue
repair. As shown in Figure 4c, at different time intervals, the tissue surrounding the gel was surgically
separated and processed by H&E staining to examine the condition of tissue damage. Basically,
no histological damage or immune response was found at any time point.
2.6. Safety Evaluation
The cytotoxicity of hydrogel mPEG45
tetrazolium (MTT) assay and hemolysis test. In Figure 5a, L929 cells treated with the highest
concentration of mPEG45
PLAla30 (100.0 ìg mL−1) for 24 h retained almost 100% viability, confirming
−
PLAla30 in vitro was evaluated by methyl thiazolyl
−
its excellent biocompatibility. Moreover, the effect of different concentrations of gel on hemolysis was
also investigated. In this part, hemolysis was measured spectroscopically according to previously
reported methods [39]. As shown in Figure 5b, no hemolysis was detected in blood samples
treated with all test concentrations of mPEG45 PLAla30, which further confirmed the outstanding
−
biocompatibility. Overall, the findings above revealed that the hydrogel showed little toxicity,
both in vitro and in vivo. Therefore, it is a promising material for usage in clinical practice.
(a)
(b)
(a) In vitro cytotoxicity of the
Figure 5.
mPEG45
Biological compatibility of mPEG45
PLAla30 toward L929 cells. Data were presented as mean
experiments of mPEG45−PLAla30. Data are presented as mean ± SD (n = 3).
−
PLAla30
.
−
±
SD (n = 5); ( ) Hemolysis
b
3. Materials and Methods
3.1. Materials
mPEG
−
OH, number-average molecular weight (Mn) = 2000 g mol−1, was purchased from
NH2 was synthesized conforming to the previously
Sigma-Aldrich (St. Louis, MO, USA). The mPEG45
−
reported protocol in our work [40]. THF and toluene were refluxed with sodium and distilled under
nitrogen before usage. N,N-Dimethylformamide (DMF) was stored over calcium hydride (CaH2) and
purified by vacuum distillation. All the other reagents and solvents were bought from Sinopharm
Chemical Reagent Co. Ltd., Beijing, China, and used as obtained.
3.2. Phase Diagram
The sol–gel transition behavior of the copolymers in PBS (pH 7.4) was determined by inverting
◦
test method with a temperature increment of 2 C per step. Samples with concentrations ranging from
◦
3.0
−
7.0 wt. % were dissolved in PBS and stirred at 0 C for 12 h. The copolymer solution (0.2 mL) was
introduced into the test tube with an inner diameter of 10.0 mm. The sol–gel transition temperature
was recorded if no flow was observed within 30 s after inverting the test tube. Each data point was the
average of three measurements.

Molecules 2018, 23, 1017
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3.3. Dynamic Mechanical Analysis
Rheological experiments were performed on a US 302 Rheometer (Anton Paar, Graz, Austria).
The copolymer solution was placed between parallel plates of 25.0 mm in diameter with a gap of
0.5 mm. To prevent the evaporation of water, the outer edge of the sandwiched sample was sealed by
a thin layer of silicon oil. The data were collected under a controlled strain
γ of 1% and a frequency of
1 rad s−1. The heating rate was 1 ◦C min−1
.
3.4. In Vitro Gel Degradation
For this, 0.5 mL of mPEG45
−
PLAla30 hydrogel was incubated in PBS (5.0 wt. %) in vials
◦
−1
(diameter = 16.0 mm) at 37 C for 10 min. PBS solutions (pH 7.4) containing 0.2 mg mL elastase K or
0.2 mg mL−1 α-chymotrypsin were used as degradation media, and hydrogels incubated in PBS were
only used as a control. Different solutions (2.0 mL) were added to the top of the gels at 37 ◦C and the
entire medium was changed daily. The weight of the remaining gel was measured daily.
3.5. In Vivo Gel Degradation
SD rats (about 180.0 g, provided by Beijing Vital River Laboratory Animal Technology Co.,
Ltd., Beijing, China) were used for the detection of gel degradation in vivo. Rats were anesthetized
by inhalation of ether before 0.5 mL of mPEG45 PLAla30 PBS solutions (5.0 wt. %) were injected
−
into the dorsal subcutaneous area of the rats using a 21-gauge needle. The rats were sacrificed on
day 7, day 14, day 21, and day 28, respectively, to monitor the degradation behavior of the gel.
The experiments on animals were carried out according to the guide for the care and use of laboratory
animals, provided by Jilin University, Changchun, China, and the procedure was approved by the
local Animal Ethics Committee.
3.6. Cytotoxicity Measurement
The relative cytotoxicity was assessed by MTT viability assay against L929 mouse fibroblasts cells.
L929 cells were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10.0% (v/v) FBS, penicillin (50.0 IU mL−1), and streptomycin (50.0 IU mL−1) at 37 ◦C in a 5.0%
(v/v) carbon dioxide atmosphere. L929 cells with a density of 6000 cells per well were planted in
96-well plates in 180.0
different concentrations (31.3–1000.0
for another 24 h before 20.0 ìL of PBS solution containing MTT (0.05 mg mL−1) was added and
incubated for a further 4 h. Then, the media was replaced with 160.0 L of dimethyl sulfoxide (DMSO).
µ
L of DMEM. After incubation for 24 h, 20.0
µL of copolymer solutions at
µ
g mL−1) were added. L929 cells were incubated with copolymers
µ
The absorbance of the solution was measured on a Bio-Rad 680 microplate reader (Hercules, CA, USA)
at 490 nm. Cell viability (%) was calculated according to the following Equation (1).
Measurements were done in five replicates.
Cell viability (%) = Asample/Acontrol × 100,
where Asample and Acontrol denote the absorbances of sample and control, respectively.
4. Conclusions
(1)
In this work, thermosensitive hydrogels mPEG45
were synthesized by ROP of L-Ala NCA monomer initiated by mPEG45
DP on the solution gel transition was investigated. It is worth noting that mPEG45
lower sol–gel transition temperature than mPEG45 PLAla22, attributed to the longer hydrophobic
segment. In addition, mPEG PLAla hydrogel had good stability and high mechanical strength
after gelation. Moreover, mPEG PLAla hydrogel did not cause any tissue damage, inflammatory
reaction, or hemolysis reaction during degradation, confirming its good biocompatibility. In summary,
−
PLAla with three different DP (14, 22, and 30)
NH2. The effect of different
PLAla30 had a
−
−
−
−
−
−

Molecules 2018, 23, 1017
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the mPEG PLAla hydrogel designed in our study had improved biocompatibility, appropriate
−
degradation, and gelation ability. Therefore, the polypeptide hydrogels showed promise for application
in tissue repair and regeneration, cell 3D culture, and treatment of cancer. Moreover, by loading
functional drugs, the hydrogels can also be used for postoperative recovery and anti-infection.
Based on the above findings, polypeptide thermosensitive hydrogels have broad prospects in the
biomedical field.
Supplementary Materials: The following are available online.
Author Contributions: W.X. and W.W. conceived and designed the experiments; J.H. performed the experiments;
J.H., X.Z. and X.F. analyzed the data; and Y.H. contributed reagents and materials. J.H. initiated and wrote this
article; X.F. helped to write and corrected the manuscript; and Y.H. discussed and suggested ideas for improvement
of this article.
Acknowledgments: This research was supported by the Key Technology Research and Development Project
of Jilin Department of Science and Technology (No. 20180201083GX) and Natural Science Foundation of Jilin
Department of Science and Technology (20170101107JC).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Ravi, M.; Paramesh, V.; Kaviya, S.R.; Anuradha, E.; Solomon, F.D. 3D cell culture systems: Advantages and
applications. J. Cell Physiol. 2015, 230, 16–26. [CrossRef] [PubMed]
Tibbitt, M.W.; Anseth, K.S. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol. Bioeng.
2009, 103, 655–663. [CrossRef] [PubMed]
Zhao, T.; Sellers, D.L.; Cheng, Y.; Horner, P.J.; Pun, S.H. Tunable, injectable hydrogels based on
peptide-cross-linked, cyclized polymer nanoparticles for neural progenitor cell delivery. Biomacromolecules
2017, 18, 2723–2731. [CrossRef] [PubMed]
4.
5.
6.
7.
Lock, L.L.; Lo, Y.; Mao, X.; Chen, H.; Staedtke, V.; Bai, R.; Ma, W.; Lin, R.; Li, Y.; Liu, G.; et al. One-component
supramolecular filament hydrogels as theranostic label-free magnetic resonance imaging agents. ACS Nano
2017, 11, 797–805. [CrossRef] [PubMed]
Deng, Y.; Yang, F.; Cocco, E.; Song, E.; Zhang, J.W.; Cui, J.J.; Mohideen, M.; Bellone, S.; Santin, A.D.;
Saltzman, W.M. Improved i.P. Drug delivery with bioadhesive nanoparticles. Proc. Natl. Acad. Sci. USA 2016
,
113, 11453–11458. [CrossRef] [PubMed]
Zhu, M.; Wei, K.; Lin, S.; Chen, X.; Wu, C.-C.; Li, G.; Bian, L. Bioadhesive polymersome for localized
and sustained drug delivery at pathological sites with harsh enzymatic and fluidic environment via
supramolecular host-guest complexation. Small 2018, 14. [CrossRef] [PubMed]
Qu, Y.; Wang, B.; Chu, B.; Liu, C.; Rong, X.; Chen, H.; Peng, J.; Qian, Z. Injectable and thermosensitive
hydrogel and pdlla electrospun nanofiber membrane composites for guided spinal fusion. ACS Appl.
Mater. Interfaces 2018, 10, 4462–4470. [CrossRef] [PubMed]
8.
9.
Zhu, C.; Lei, H.; Fan, D.; Duan, Z.; Li, X.; Li, Y.; Cao, J.; Wang, S.; Yu, Y. Novel enzymatic crosslinked hydrogels
that mimic extracellular matrix for skin wound healing. J. Mater. Sci. 2018, 53, 5909–5928. [CrossRef]
Shahriari, D.; Shibayama, M.; Lynam, D.A.; Wolf, K.J.; Kubota, G.; Koffler, J.Y.; Tuszynski, M.H.;
Campana, W.M.; Sakamoto, J.S. Peripheral nerve growth within a hydrogel microchannel scaffold supported
by a kink-resistant conduit. J. Biomed. Mater. Res. Part A 2017, 105, 3392–3399. [CrossRef] [PubMed]
10. Xu, H.-L.; Tian, F.-R.; Lu, C.-T.; Xu, J.; Fan, Z.-L.; Yang, J.-J.; Chen, P.-P.; Huang, Y.-D.; Xiao, J.; Zhao, Y.-Z.
Thermo-sensitive hydrogels combined with decellularised matrix deliver bfgf for the functional recovery of
rats after a spinal cord injury. Sci. Rep. 2016, 6, 38332. [CrossRef] [PubMed]
11. Lu, D.; Li, Y.; Li, T.; Zhang, Y.; Dou, F.; Wang, X.; Zhao, X.; Ma, H.; Guan, X.; Wei, Q.; et al. Surgical
adhesive: Synthesis and properties of thermoresponsive pluronic l-31-3,4-dihydroxyphenylalanine-arginine
derivatives. J. Appl. Polym. Sci. 2017, 134. [CrossRef]
12. Li, P.; Zhang, J.; Dong, C.-M. Photosensitive poly(o-nitrobenzyloxycarbonyl-L-lysine)-b-peo polypeptide
copolymers: Synthesis, multiple self-assembly behaviors, and the photo/ph-thermo-sensitive hydrogels.
Polym. Chem. 2017, 8, 7033–7043. [CrossRef]

Molecules 2018, 23, 1017
10 of 11
13. Cui, H.; Shao, J.; Wang, Y.; Zhang, P.; Chen, X.; Wei, Y. PLA-PEG-PLA and its electroactive tetraaniline
copolymer as multi-interactive injectable hydrogels for tissue engineering. Biomacromolecules 2013, 14,
1904–1912. [CrossRef] [PubMed]
14. Wang, D.K.; Varanasi, S.; Strounina, E.; Hill, D.J.T.; Symons, A.L.; Whittaker, A.K.; Rasoul, F. Synthesis and
characterization of a POSS-PEG macromonomer and POSS-PEG-PLA hydrogels for periodontal applications.
Biomacromolecules 2014, 15, 666–679. [CrossRef] [PubMed]
15. Michlovska, L.; Vojtova, L.; Humpa, O.; Kucerik, J.; Zidek, J.; Jancar, J. Hydrolytic stability of end-linked
hydrogels from PLGA-PEG-PLGA macromonomers terminated by alpha,omega-itaconyl groups. RSC Adv.
2016, 6, 16808–16816. [CrossRef]
16. Wang, P.; Chu, W.; Zhuo, X.; Zhang, Y.; Gou, J.; Ren, T.; He, H.; Yin, T.; Tang, X. Modified PLGA-PEG-PLGA
thermosensitive hydrogels with suitable thermosensitivity and properties for use in a drug delivery system.
J. Mater. Chem. B 2017, 5, 1551–1565. [CrossRef]
17. Zhang, Y.; Zhang, J.; Chang, F.; Xu, W.; Ding, J. Repair of full-thickness articular cartilage defect using stem
cell-encapsulated thermogel. Mater. Sci. Eng. C 2018, 88, 79–87. [CrossRef] [PubMed]
18. Dong, X.; Chen, H.; Qin, J.; Wei, C.; Liang, J.; Liu, T.; Kong, D.; Lv, F. Thermosensitive porphyrin-incorporated
hydrogel with four-arm peg-pcl copolymer (ii): Doxorubicin loaded hydrogel as a dual fluorescent drug
delivery system for simultaneous imaging tracking in vivo. Drug. Deliv. 2017, 24, 641–650. [CrossRef]
[PubMed]
19. Luo, Z.; Jin, L.; Xu, L.; Zhang, Z.L.; Yu, J.; Shi, S.; Li, X.; Chen, H. Thermosensitive PEG-PCL-PEG (PECE)
hydrogel as an in situ gelling system for ocular drug delivery of diclofenac sodium. Drug. Deliv. 2016, 23,
63–68. [CrossRef] [PubMed]
20. Wang, S.-J.; Zhang, Z.-Z.; Jiang, D.; Qi, Y.-S.; Wang, H.-J.; Zhang, J.-Y.; Ding, J.-X.; Yu, J.-K. Thermogel-coated
poly(epsilon-caprolactone) composite scaffold for enhanced cartilage tissue engineering. Polymers 2016
8, 200. [CrossRef]
,
21. Navath, R.S.; Menjoge, A.R.; Dai, H.; Romero, R.; Kannan, S.; Kannan, R.M. Injectable PAMAM
dendrimer-PEG hydrogels for the treatment of genital infections: Formulation and in vitro and in vivo
evaluation. Mol. Pharm. 2011, 8, 1209–1223. [CrossRef] [PubMed]
22. Moon, H.J.; Choi, B.G.; Park, M.H.; Joo, M.K.; Jeong, B. Enzymatically degradable thermogelling
poly(alanine-co-leucine)-poloxamer-poly(alanine-co-leucine). Biomacromolecules 2011, 12, 1234–1242.
[CrossRef] [PubMed]
23. Zheng, Y.; Cheng, Y.; Chen, J.; Ding, J.; Li, M.; Li, C.; Wang, J.-C.; Chen, X. Injectable hydrogel-microsphere
construct with sequential degradation for locally synergistic chemotherapy. ACS Appl. Mater. Interfaces 2017
,
9, 3487–3496. [CrossRef] [PubMed]
24. Cheng, Y.; He, C.; Ding, J.; Xiao, C.; Zhuang, X.; Chen, X. Thermosensitive hydrogels based on polypeptides
for localized and sustained delivery of anticancer drugs. Biomaterials 2013, 34, 10338–10347. [CrossRef]
[PubMed]
25. Conde, J.; Oliva, N.; Atilano, M.; Song, H.S.; Artzi, N. Self-assembled RNA-triple-helix hydrogel scaffold for
microRNA modulation in the tumour microenvironment. Nat. Mater. 2016, 15, 353. [CrossRef] [PubMed]
26. Li, X.; Ding, J.; Zhang, Z.; Yang, M.; Yu, J.; Wang, J.; Chang, F.; Chen, X. Kartogenin-incorporated thermogel
supports stem cells for significant cartilage regeneration. ACS Appl. Mater. Interfaces 2016, 8, 5148–5159.
[CrossRef] [PubMed]
27. Zhang, Y.; Ding, J.; Sun, D.; Sun, H.; Zhuang, X.; Chang, F.; Wang, J.; Chen, X. Thermogel-mediated sustained
drug delivery for in situ malignancy chemotherapy. Mater. Sci. Eng. C 2015, 49, 262–268. [CrossRef]
[PubMed]
28. Zhang, Y.-B.; Ding, J.-X.; Xu, W.-G.; Wu, J.; Chang, F.; Zhuang, X.-L.; Chen, X.-S.; Wang, J.-C. Biodegradable
thermogel as culture matrix of bone marrow mesenchymal stem cells for potential cartilage tissue engineering.
Chin. J. Polym. Sci. 2014, 32, 1590–1601. [CrossRef]
29. Sitoci-Ficici, K.H.; Matyash, M.; Uckermann, O.; Galli, R.; Leipnitz, E.; Later, R.; Ikonomidou, C.; Gelinsky, M.;
Schackert, G.; Kirsch, M. Non-functionalized soft alginate hydrogel promotes locomotor recovery after spinal
cord injury in a rat hemimyelonectomy model. Acta Neurochir. 2018, 160, 449–457. [CrossRef] [PubMed]
30. Ansari, S.; Diniz, I.M.; Chen, C.; Sarrion, P.; Tamayol, A.; Wu, B.M.; Moshaverinia, A. Human periodontal
ligament- and gingiva-derived mesenchymal stem cells promote nerve regeneration when encapsulated in
alginate/hyaluronic acid 3D scaffold. Adv. Healthc. Mater. 2017, 6. [CrossRef] [PubMed]

Molecules 2018, 23, 1017
11 of 11
31. Liu, H.; Ding, J.; Li, C.; Wang, C.; Wang, Y.; Wang, J.; Chang, F. Hydrogel is superior to fibrin gel as matrix of
stem cells in alleviating antigen-induced arthritis. Polymers 2016, 8, 182. [CrossRef]
32. Chaudhuri, O.; Gu, L.; Klumpers, D.; Darnell, M.; Bencherif, S.A.; Weaver, J.C.; Huebsch, N.; Lee, H.-P.;
Lippens, E.; Duda, G.N.; et al. Hydrogels with tunable stress relaxation regulate stem cell fate and activity.
Nat. Mater. 2016, 15, 326. [CrossRef] [PubMed]
33. Knight, E.; Przyborski, S. Advances in 3D cell culture technologies enabling tissue-like structures to be
created in vitro. J. Anat. 2015, 227, 746–756. [CrossRef] [PubMed]
34. Fu, S.; Ni, P.; Wang, B.; Chu, B.; Zheng, L.; Luo, F.; Luo, J.; Qian, Z. Injectable and thermo-sensitive
PEG-PCL-PEG copolymer/collagen/n-ha hydrogel composite for guided bone regeneration. Biomaterials
2012, 33, 4801–4809. [CrossRef] [PubMed]
35. Yuan, B.; He, C.; Dong, X.; Wang, J.; Gao, Z.; Wang, Q.; Tian, H.; Chen, X. 5-fluorouracil loaded
thermosensitive PLGA-PEG-PLGA hydrogels for the prevention of postoperative tendon adhesion. RSC Adv.
2015, 5, 25295–25303. [CrossRef]
36. Wei, L.; Chen, J.; Zhao, S.; Ding, J.; Chen, X. Thermo-sensitive polypeptide hydrogel for locally sequential
delivery of two-pronged antitumor drugs. Acta Biomater. 2017, 58, 44–53. [CrossRef] [PubMed]
37. Choi, Y.Y.; Jang, J.H.; Park, M.H.; Choi, B.G.; Chi, B.; Jeong, B. Block length affects secondary
structure, nanoassembly and thermosensitivity of poly(ethylene glycol)-poly(L-alanine) block copolymers.
J. Mater. Chem. 2010, 20, 3416–3421. [CrossRef]
38. Cheng, Y.; He, C.; Xiao, C.; Ding, J.; Zhuang, X.; Huang, Y.; Chen, X. Decisive role of hydrophobic side groups
of polypeptides in thermosensitive gelation. Biomacromolecules 2012, 13, 2053–2059. [CrossRef] [PubMed]
39. Xu, W.; Ding, J.; Xiao, C.; Li, L.; Zhuang, X.; Chen, X. Versatile preparation of intracellular-acidity-sensitive
oxime-linked polysaccharide-doxorubicin conjugate for malignancy therapeutic. Biomaterials 2015, 54, 72–86.
[CrossRef] [PubMed]
40. Ding, J.; Shi, F.; Xiao, C.; Lin, L.; Chen, L.; He, C.; Zhuang, X.; Chen, X. One-step preparation of
reduction-responsive poly(ethylene glycol)-poly(amino acid)s nanogels as efficient intracellular drug delivery
platforms. Polym. Chem. 2011, 2, 2857–2864. [CrossRef]
Sample Availability: Samples of the compounds are available from the authors.
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