A photo-degradable injectable self-healing hydrogel based on star poly(ethylene glycol)-: B -polypeptide as a potential pharmaceuticals delivery carrier

Article abstract of DOI:10.1039/c8sm01575a

As one of the most promising biomaterials, injectable self-healing hydrogels have found broad applications in a number of fields such as local drug delivery. However, controlled release of drugs in hydrogels is still difficult to realize up to now. Here, we report a novel photo-degradable injectable self-healing hydrogel based on the hydrophobic interaction of a biocompatible four-arms star polymer, poly(ethylene glycol)-b-poly(γ-o-nitrobenzyl-l-glutamate). The hydrophobic interaction between poly(γ-o-nitrobenzyl-l-glutamate) not only connects poly(ethylene glycol)-b-poly(γ-o-nitrobenzyl-l-glutamate) together with a crosslink but also provides a hydrophobic domain to encapsulate hydrophobic pharmaceuticals such as doxorubicin (DOX). Due to the dynamic character of the hydrophobic interaction, the hydrogel exhibits excellent injectable and self-healing ability. In particular, the photolabile o-nitribenzyl ester group is cleaved under UV irradiation. As a result, the hydrophobic domain transforms into the hydrophilic one and the embedded DOX is released effectively. An increasing release ratio of DOX dramatically enhances the apoptosis ratio of HeLa cells. We expect these attractive properties may be beneficial to practical applications of the hydrogel as an effective local drug delivery means in a truly physiological environment.

Full text of DOI:10.1039/c8sm01575a

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ISSN 1744-683X
PAPER
Jure Dobnikar et al.
Rational design of molecularly imprinted polymers
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A photo-degradable injectable self-healing hydrogel based on star
poly(ethylene glycol)-b-polypeptide as potential pharmaceuticals
delivery carrier
intReceived 00th January 20xx,
Accepted 00th January 20xx
Dinglei Zhao,a Quan Tang,a Qiang Zhou,a Kang Peng,a Haiyang Yang*,a and Xingyuan Zhang*a
DOI: 10.1039/x0xx00000x
As one of the most promising biomaterials, injectable self-healing hydrogels have found broad applications in a number of
fields such as local drugs delivery. However, the controlled release of drugs in hydrogels is still difficult to realize up to
now. Here, we report a novel photo-degradable injectable self-healing hydrogel based on the hydrophobic interaction of a
biocompatible four-arms star polymer, poly(ethylene glycol)-b-poly(γ-o-nitrobenzyl-L-glutamate). The hydrophobic
interaction between poly(γ-o-nitrobenzyl-L-glutamate) not only connects poly(ethylene glycol)-b-poly(γ-o-nitrobenzyl-L-
glutamate) together as a crosslink but also provides a hydrophobic domain to encapsulate hydrophobic pharmaceuticals
such as Doxorubicin (DOX). Due to the dynamic character of the hydrophobic interaction, the hydrogel exhibits excellent
injectable and self-healing ability. In particular, the photolabile o-nitribenzyl ester group is cleaved under UV irradiation. As
a result, the hydrophobic domain transforms into the hydrophilic one and the DOX embedded is released effectively. The
increasing release ratio of Dox dramatically enhances the apoptosis ratio of HeLa cell. We expect these attractive
properties maybe beneficial to the practical application of hydrogel as an effective local drugs delivery in a truly
physiological environment.
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Introduction
Hydrogels are a sort of attractive soft materials1, 2 because of many injectable self-healing hydrogels have been designed in
their considerable practical potential for biomedical and drug delivery, the release mechanism of the carriers is passive
pharmaceutical applications3-5. In particular, injectable self- material degradation. Therefore, it is still a challenge to active
healing hydrogels5-12, which can be implanted through a control the release of drugs on demand in injectable self-
syringe at the targeted sites and then rapidly form bulk gels in healing hydrogels.
situ, have been paid much attention in recent researches.
Active controlling release can efficiently optimize the
To date, many works about 3D injectable self-healing pharmacokinetic of drugs and reduce the amount of necessary
hydrogels have been reported. The methods of fabricating drugs. The convenience of fewer and more effective doses also
injectable self-healing hydrogels are generally categorized into increases patient compliance.26 Stimuli-responsive hydrogels
three types: physical cross-linking,13-18 dynamic chemical cross have advantages in realizing on demand controlled release of
28, 29
-linking19-22 or the dual mechanism.19, 23 Among all methods, payloads.27 A variety of stimuli including pH,4,
redox
physical cross-linking is the most commonly used due to the species,4 photo-irradiation,30 enzymes31 have been applied to
dynamical self-adaptation. For instance, Leong et al.24 reported accomplish on demand release of payloads. Among all stimuli,
an injectable hydrogel formed from poly(ethylene oxide)s and light is of particular fascinating stimulus to manipulate the
α-cyclodextrin for drug delivery. The hydrogel formation was properties of hydrogels as it is a remote stimulus that can be
based on physical crosslinking induced by supramolecular self- controlled spatially and temporally with great ease and
assembling. Pochan et al.25 demonstrated an injectable convenience.32-39 Hence, photo responsive injectable self-
hydrogel based on curcumin-loaded β-sheet assembling healing hydrogels are ideal on demand release carriers.
peptide nanofiber hydrogels for local chemotherapy. Although In recent years, biodegradable polypeptides have been
proposed to design stimuli-responsive injectable hydrogels,
due to their hierarchical self-assembly,40 multiple secondary,41
biocompatibility42 and biodegradability.43 Deming et al.44
reported a photo-degradable copolypeptide hydrogel which
included charged amino acid residues for better water
solubility. The charges, especially positive charges, may cause
inevitable side effects such as unfavourable protein absorption
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in vivo. Hence, non-ionic poly(ethylene glycol) (PEG) is a (Shanghai, China). All other reagents were purchased from
superior choice as hydrophilic block, especially 4-arm PEG Sinopharm Chemical Reagent Co Ltd. and used as received.
which has attracted extensive attention due to its perfect Water was deionized with a Milli-Q SPreagent water system
spatial structure,21, 45, 46 dissolubility and biocompatibility.47
Here, we designed a photo-degradable injectable self-healing 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were
hydrogel based on the hydrophobic interaction of acquired on a 300 MHz Bruker instrument. ESI-MS experiment
biocompatible four-arms star polymer, poly(ethylene glycol)-b- was performed on Thermo Scientific LTQ Orbitrap Mass
poly(γ-o-nitrobenzyl-L-glutamate). The hydrophobic Spectrometer equipped with an electrospray interface. Fourier
(Millipore) to a specific resistivity of 18.4 mΩ cm.
a
interaction between poly(γ-o-nitrobenzyl-L-glutamate) not transform infrared (FT-IR) spectra were recorded on a Bruker
only connects poly(ethylene glycol)-b-poly(γ-o-nitrobenzyl-L- VECTOR-22 IR spectrometer. Molecular weight and molecular
glutamate) together as a reversible crosslink but also provides weight distributions, PDI, were determined by gel permeation
a
hydrophobic domain to encapsulate hydrophobic chromatography (GPC) equipped with a Waters 1515 pump
pharmaceuticals such as Doxorubicin (DOX). Upon irradiation, and a Waters 2414 differential refractive index detector (set at
the full cleavage of o-nitrobenzyl ester was observed, resulting 30°C). It used a series of three linear Styragel columns at an
in the hydrophobic crosslinks disruption and release of oven temperature of 45 °C. The eluent was DMF at a flow rate
encapsulated Doxorubicin. The increasing release ratio of of 1.0 mL/min. A series of low polydispersity polystyrene
hydrophobic pharmaceutical Dox due to hydrophobic standards were employed for calibration. The degrees of
microstructure disintegration upon irradiation enhanced polymerization, DPs, ware determined by 1H NMR analysis.
apoptosis ratio of HeLa cell. With these attractive properties, UV/vis absorbance was conducted on a double-beam UV-Vis
the photo-responsive injectable hydrogel as presented here spectrophotometer (Shimadzu). 365nm LED lamp was used for
may provide new opportunities in
a truly physiological UV irradiation. Fluorescence experiments were conducted on a
environment for site-specific on demand drug release.
RF-5301/PC (Shimadzu) fluorospectro photometer. Rheological
properties of all prepared hydrogels were studied on a
Rheometer (TA instruments, AR-G2) with a platform of 40 mm
diameter.
Experiment
Synthetic procedure
Materials and methods
Schematic synthetic procedures are shown in scheme 2, Figure
p-nitrophenylchloroformate was purchased from Meyer
chemical Co Ltd without further purity. L-Glutamate acid
(Aladdin) was used as received. 1,1,3,3-tetramethylguanidine
(TMG, 98%, Aldrich) were distilled under reduced pressure.
Tetra-PEG-NH2 was purchased from Sigma-Aldrich. Dox·HCl
and Nile Red were from J&K chemical. N,N-dimethylacetamide
(extra dry, with molecular sieves, water≤50 ppm) and o-
nitrobenzyl alcohol were purchased from Energy Chemical. The
HeLa cells were purchased from the Shanghai Institute of Cell
Biology (Shanghai, China). Fetal bovine serum (FBS), 4,6-
diamidino-2-phenylindole (DAPI), trypsin, phosphate buffered
saline (PBS), and Dulbecco’s modified Eagle’s medium (DMEM)
were purchased from GIBCO and used as received. Cell culture
lysis buffer, 4’,4’,6-diamidino-2-phenylindole bromide (MTT)
were purchased from Beyotime Institute of Biotechnology
S1.
Synthesis of the N-(p-nitrophenoxycarbonyl)-γ-o-nitrobenzyl-L-
glutamate (NPNBLG)
NPNBLG was synthesised according to the literature.41 Briefly,
o-nitrobenzyl alcohol (1.53 g, 10 mmol, 1.0 equiv.) was
dissolved in dichloromethane (30 mL), then phosphorus
tribromide (1.05 mL, 1.1 equiv) was added at ice bath. The
mixture was then stirred at room temperature and monitored
by TLC for 6 h. After that, the reaction mixture was washed
with saturated brine, dried over MgSO4, and the solvent was
removed under reduced pressure. Then the product was dried
overnight in a vacuum oven at room temperature, affording o-
nitrobenzyl bromide as a yellowish solid for using without
further purification.
Scheme 1: Schematic illustration of the design and application of the photo-responsive injectable hydrogel.
2 | J. Name., 2012, 00, 1-3
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In
a
250-mL
round
bottom
flask,
N,N,N’,N’- polydispersity index (PDI, Mw/Mn) was quantified by gel
tetramethylguanidine (11 mL, 87 mmol) was added slowly to a permeation chromatography.
Hydrogel preparation
stirred L-glutamic acid and L-glutamic acid copper (II) complex
(30.8 g, 86 mmol) in DMF (40 mL) and distilled water (6 mL).
The mixture turned to dark blue. After all solids were dissolved
(~2h), additional DMF (31 mL) was added. Then, o-nitrobenzyl
bromide (25 g, 90 mmol) was added to the above solution in
one portion. The reaction solution became darker and was
kept at 40 °C for 38 h. After that, acetone (500 mL) was added
to the mixture and stirred until a fine precipitate was obtained
(~1 h). The violet solid was collected by filtration, followed by
addition of freshly prepared EDTA (20 g)/sodium bicarbonate
(10 g) aqueous solution (150 mL) and further stirring for 24 h.
The product was collected by filtration and washed with DI
water. Further purification was performed by recrystallizing
The hydrogel was prepared by dispersing star PEG-polypeptide
conjugates in water. The obtained solutions were gently
heated and vigorous stirred for a few minutes to ensure
complete dissolution. The solutions were then cooled to room
temperature to allow gelation.
Rheological measurements of the hydrogels
Rheological properties of all prepared hydrogels were studied
on a Rheometer (TA instruments, AR-G2) with a platform of
40mm diameter. The frequency-sweep measurement was
conducted in a constant-strain (0.5%) mode over the
frequency range of 0.1-100 rad·s-1 at 37°C. Before study the
self-healing property, a strain-sweep measurement was
conducted at a constant frequency of 6.283 rad·s-1 over the
strain range of 0.1% to 2000% at 37°C. Finally, a strain step
cycled between 1% and 800% was performed at 37°C and
6.283 rad·s-1.
1
from H2O/isopropanol. Obtained 15.1 g (yield: 49%) H NMR
[D2O/DCl (1wt%), δ, ppm]: 8.03 (d, 1H), 7.65 (t, 1H), 7.48~7.55
(m, 2H), 5.39 (s, 2H), 4.05 (t, 1H), 2.65 (t, 2H), 2.18 (m, 2H).
To a solution of γ-o-nitrobenzyl-L-glutamate (2.82 g, 10 mmol)
in THF (50 mL), p-nitrophenylchloroformate (2.03 g, 10 mmol)
was added at room temperature and the mixture was stirred
at 40 °C for 24 h. The solvent was removed under reduced
pressure, 50 mL DCM was added. Then the solution was
washed with distilled water and a saturated NaCl aqueous
solution, dried over anhydrous MgSO4, filtered and
concentrated by a rotary evaporator. The resulting solid
residue was fractionated by column chromatography using a
mixed solvent of ethyl acetate/n-hexane (1/1 v/v) to obtain
NPNBLG as a white solid (1.6 g, Yield: 35%) 1H NMR [CD3OD, δ,
ppm]: 8.26 (d, 2H), 8.11 (d, 1H), 7.70-7.60 (m, 3H), 5.52 (s, 2H),
4.32 (m, 1H), 2.65 (t, 2H), 2.35 (m, 1H) 2.10 (m, 1H).
The gel to sol transition upon UV light
The frequency-sweep measurement of hydrogel before and
after 30 min UV irradiation was carried out to study its light-
sensitive gel-sol transition.
Self-assembly of star PEG-polypeptide conjugates and UV-vis
absorbance spectra upon UV light
2 mg star PEG-polypeptide conjugates was dissolved in 1 mL
1,4-dioxane, stirred and maintained at a predetermined
temperature in a water bath for 25 min. Then, 8 mL PBS buffer
(pH 7.4, 10 mM) was slowly injected at an addition rate of 2
mL/h via a syringe pump. After stirring for another 2 h, 1,4-
dioxane was removed by dialysis (cellulose membrane,
MWCO: 3.5 kDa) against PBS buffer (pH 7.4, 10 mM). Fresh
PBS buffer was replaced approximately every ~4 h. The UV-vis
evolution of micelles upon UV irradiation (1 mW/cm2) was
recorded at predetermined time intervals.
Synthesis of the N-(p-nitrophenoxycarbonyl)-γ-benzyl-L-glutamate
(NPBLG)
To a solution of γ-benzyl-L-glutamate (2.37 g, 10 mmol) in THF
(50 mL), p-nitrophenyl chloroformate (2.03 g, 10 mmol) was
added at room temperature and the mixture was stirred at 40
°C for 24 h. The resulting mixture was washed with distilled
water and a saturated NaCl aqueous solution, dried over
anhydrous MgSO4, filtered, and concentrated by a rotary
evaporator. The resulting solid residue was fractionated by
column chromatography using a mixed solvent of ethyl
acetate/n-hexane (1/1 v/v) to obtain NPBLG as a white solid
Preparation of NR-loaded star PEG-polypeptide conjugates micelle
and light-triggered release of Nile Red
2 mg star PEG-polypeptide conjugates and 20 μg Nile Red were
dissolved in 1 mL 1,4-dioxane , stirred and maintained at a
predetermined temperature in a water bath for 25 min. Then,
8 mL PBS buffer (pH 7.4, 10 mM) was slowly injected at an
addition rate of 2 mL/h via a syringe pump. After stirring for
another 2 h, 1,4-dioxane was removed by dialysis (cellulose
membrane, MWCO: 3.5 kDa) against PBS buffer (pH 7.4, 10
mM). Fresh PBS buffer was replaced approximately every ~4 h.
The fluorescence evolution of NR-loaded micelles upon UV
irradiation (1 mW/cm2) was recorded at predetermined time
1
(1.4 g, Yield: 35%). H-NMR [CD3OD, δ, ppm]: 2.13–2.45 (m,
2H), 2.62 (m, 2H), 4.48–4.56 (m, 1H),F 5.16 (s, 2H), 6.02 (d,
1H), 7.30–7.34 (m, 2H), 7.37 (m, 5H), 8.25 (m, 2H).
Synthetic and characterization of the polymer
Typically, a solution of NPNBLG (0.448 g
， 1.0 mmol) and
intervals (λex = 550 nm)
.
tetra-PEG-NH2(0.4 g, 0.04 mmol) in N,N-dimethylacetamide (1
mL) was stirred at 60 °C for 48 h under nitrogen. The reaction
mixture was poured into an excess of ether for three times,
and dried in vacuum oven at room temperature, yielding a
solid powder star poly(ethylene glycol)-b-poly(γ-o-nitrobenzyl-
L-glutamate) (0.52 g, Yield: 74%).
Preparation of DOX-loaded hydrogel and controlled released of
DOX payload
Typically, 30 mg DOX·HCl was dissolved in 1 mL DMSO and 2
mL triethylamine was then added and stirred at room
temperature overnight. After that, 300 mg polymer was added
and stirred at ambient temperature for 2 h. The mixtures were
then subjected to dialysis against PBS buffer (pH 7.4, 10 mM)
for 24 h to remove unloaded DOX and DMSO solvent. The
Monomer conversion and mean degree of polymerization (DP)
were determined by 1H NMR. Number-average molecular
weight (Mn) was determined by 1H NMR analysis, and
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dialysate was adjusted to predetermined volumes (pH 7.4, 10
mM) by adding PBS buffer. According to a standard calibration
curve, the DOX loading efficiencies (LE) and the DOX loading
contents (LC) were estimated. For the triggered release of
DOX, in a typical release experiment, DOX-loaded hydrogels
(500 μL) were transferred to a dialysis cell with a molecular
weight membrane (MWCO: 3.5 kDa) and then dialyzed against
9.5 mL of PBS buffer (pH 7.4, 10 mM) at 37°C.
In Vitro Cytotoxicity Measurement.
Cell viability was examined by the MTT assay. HeLa cells were
seeded in 96-well plate at a density of 104 cells per well in 100
μL DMEM medium with 10% FBS at 37 °C under 5% CO2
humidified atmosphere. Drug-loaded micelles with or without
30 min UV irradiation (1 mW/cm2) were then added to target a
final concentration of 3.0 g/L. After incubating for 24 h, MTT
reagent (in 20 μL PBS buffer, 5 mg/mL) was added to each
well, and the cells were further incubated with 5% CO2 for 4 h
at 37 °C. The culture medium in each well was removed and
replaced by 150 μL of DMSO. The solution from each well was
transferred to another 96-well plate, and the absorbance
values were recorded at a wavelength of 490 nm by a
microplate reader (Thermo Fisher). The cell viability is
calculated to be A490,treated/A490,control × 100%, where A490,treated
and A490,control are the absorbance values in the presence and
absence of polymeric micelles, respectively. Each experiment
was done in quadruple, and the data were shown as the mean
value.
Figure 1: (a) 1H NMR of γ-o-nitrobenzyl-L-glutamate in D2O with a drop of DCl. (b) 1H
NMR of N-(p-nitrophenoxycarbonyl)-γ-o-nitrobenzyl-L-glutamate in CD3OD. (c) 1H NMR
of amphiphilic diblock copolymers t-P56-b-NG5 in CDCl3. d) The GPC trace recorded for
amphiphilic diblock copolymer t-P56-b-NG5 by DMF elution.
Table 1: The structural parameters of star PEG-polypeptide conjugates.
Star PEG-polypeptide
t-PEG56-b-PNBLG5
t-PEG56-b-PNBLG7
t-PEG56-b-PNBLG10
t-PEG56-b-PNBLG12
t-PEG56-b-PNBLG15
abbreviation
t-P56-b-NG5
t-P56-b-NG7
t-P56-b-NG10
t-P56-b-NG12
t-P56-b-NG15
Mn/kDaa
Mw/Mnb
15.6
1.07
17.9
1.05
21.3
1.03
23.5
1.08
26.9
1.07
aDetermined by 1H NMR analysis. bObtained from GPC analysis using DMF as eluent.
Determination of apoptotic cell death
The star PEG-polypeptide conjugates were synthesised using a
novel method according to previously reported work.48 As
shown in Scheme 2, N-(p-nitrophenoxycarbonyl)-γ-o-
nitrobenzyl-L-glutamate, which was prepared by N-
carbamoylation of γ-o-nitrobenzyl-L-glutamate(Figure 1a and
1b), underwent successful polycondensations with tetra-PEG-
NH2 as initiator to obtain a solid powder star poly(ethylene
glycol)-b-poly(γ-o-nitrobenzyl-L-glutamate), PEG-polypeptide
conjugates.
The apoptotic cell death induced by Dox-loaded micelles was
examined via DAPI staining of HeLa cells cultured in glass-
bottom petri dishes. After treating with and without
irradiation Dox-loaded micelles for 24 h, the cells were mixed
with DAPI staining solution for each dish. After further
incubation for 20 min, the staining solution was removed and
washed three times with PBS buffer media. Cell nuclei were
stained by DAPI and observed under a confocal laser scanning
microscopy using a 405 nm laser. The emission detection
channel was set at 440-480 nm.
A series of PEG-polypeptide conjugates were synthesized.
Monomer conversion and mean degree of polymerization (DP)
were determined by 1H NMR from the ratio of the
characteristics of o-nitrobenzyl ester moiety at 5.5 ppm and
the characteristics of the PEG moiety at 3.5 ppm (Figure 1c and
Figure S4). Number-average molecular weight (Mn) was
determined by 1H NMR analysis, and polydispersity index (PDI)
was quantified by gel permeation chromatography (Figure 1d
and Figure S5). A summary table was shown in Table 1.
Rheological characterization of hydrogel
Results and discussion
Synthesis and characterization of star PEG-polypeptide conjugates
Table2: The mechanical properties of hydrogel of different composition and
concentrations.
Weight percent/%
Entry
10
20
30
G’/kPa
0.17
0.55
0.71
0.85
0.96
G’’/kPa
0.19
G’/kPa
0.85
G’’/kPa
0.53
G’/kPa
1.76
G’’/kPa
0.77
t-P56-b-NG5
t-P56-b-NG7
t-P56-b-NG10
t-P56-b-NG12
t-P56-b-NG15
0.31
4.33
0.93
7.25
1.18
0.25
6.30
1.35
11.01
15.46
19.93
1.65
0.28
8.60
2.42
2.86
0.28
10.49
2.65
4.89
Scheme 2: The synthetic route of star PEG-polypeptide conjugates was shown.
The mechanical properties of hydrogel were carried out on a rotated rheometer.
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formed elastic gels over a broad frequency range except 10 wt%
t-P56-b-NG5 sample, likely because low hydrophobic content in
this sample could not support its weight. The storage modulus
and loss modulus of hydrogels increased with increasing star
PEG-polypeptide conjugates concentration (Table 2), following
trends similar to the previous report.44 And the corresponding
intuitive results were shown in Figure 2.
The mechanical properties of hydrogel similar to surrounding
tissue can maintain the integrity of hydrogel under tissue
squeezing. The storage modulus of diseased mammalian
tissues and organs, such as premalignant breast and fibrotic
liver was usual in ~1.6 kPa~2.2 kPa.49 Therefore, we chose 30
wt% t-P56-b-NG5 hydrogel for the next experiments due to
approximate storage modulus.
Self-healing and injectable properties of hydrogels
Hydrogels with
a self-healing ability will extend thier
serviceable range and lifespan. To investigate the mechanical
properties regarding shear-thinning and recovery, we
performed rheological tests on 30 wt% t-P56-b-NG5 hydrogel. A
strain sweep measurement was conducted first to figure out
the linear viscoelastic region and the gel-sol transition point.
As shown in Figure 3a, storage modulus (G’) and loss modulus
(G’’) remained constant during strain increase from 0.1% to
400%, and the value of G’ was much larger than that of G’’,
suggesting that the hydrogel could withstand the deformation
at 37 °C. While the strain kept increasing beyond 400%, both G’
and G’’ dramatically went down, and showed a crossover at
strain γ=410%. This indicated that severe slippage of polymer
chains appeared and the hydrogel network was disrupted into
a sol state. This excellent shear-thinning property of the
prepared hydrogel makes it possible to be injected with a
springe (Figure 3a inset).
Figure 2: Rheology of star PEG-polypeptide conjugates of hydrogel. (a) Storage modulus
(G’/kPa) and (b) Loss modulus (G’’/kPa) of hydrogel at different composition and
concentration.
To quantify the mechanical properties of star PEG-polypeptide
conjugates, a series of hydrogels with different compositions
and concentrations were prepared. The frequency sweep
measurement of them was performed (Figure S7). All samples
Figure 3: (a) Strain-sweep measurements of a 30 wt% t-P56-b-NG5 hydrogel at 37 °C (inset: injection test of the hydrogel stained with Eosin Y at room temperature). (b) Repeated
dynamic strain step tests (γ=1 % or 800 %). (c) Separated pieces of hydrogel (stained with Eosin Y and without dye, respectively) were brought together and then self-healing
process occurred within 5 min, and the healed hydrogel could support its own weight.
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Then, the self-healing was measured by repeated dynamic
strain step tests (γ=1% or 800%). The results were shown in
Figure 3b, when subjected to 800% strain, the G’ of the
hydrogel dropped from 2.5 kPa to 0.1 kPa immediately and
became smaller than G’’ in value, indicating that the hydrogel
was disrupted and subsequently transformed into a sol state.
After decreasing the strain back to 1%, G’ and G’’ almost
recovered immediately to their initial values, which
demonstrated the self-healing capability of the hydrogel. The
recovery behavior was relatively fast and completely reversible
over the cyclic tests. The self-healing ability of the hydrogel
was further demonstrated visually in Figure 3c. A uniform
sample was cut into two pieces (one stained with Eosin Y, the
other without), and after putting back together, these two
hydrogel fragments could adhere and self-repair into one
integral piece.
Figure 4: Photographs of the reversed vial test before (a) and after (b) 30 min UV
irradiation at 37°C. Frequency-sweep measurement of hydrogel was performed before
(c) and after (d) 30 min UV irradiation at 37°C.
Photograph and rheological studies on gel to sol upon UV
irradiation
Spectroscopy analysis
The photo degradation of the freshly prepared hydrogel in vial
was researched. Before UV irradiation, hydrogel in vial
couldn’t flow (Figure 4a). And the frequency-sweep
measurement of hydrogel shown(Figure 4c) that G’ was larger
than G’’, indicating the sample was in gel state. After 30 min
UV irradiation, the gel degraded to a fluidic solution (Figure 4b).
Meanwhile, the colour of the hydrogel transformed from pale
yellow to dark red. The frequency-sweep of the sample after
UV irradiation was shown in Figure 4d, G’<G” over the entire
frequency range, suggesting the sample was in sol state. The
value of G’ and G’’ after UV irradiation decreased sharply
(~90%), indicating that original interaction between chains had
been destroyed.
To verify the degradation mechanism of hydrogel upon UV
irradiation, the aqueous micellar solution (0.2 g/L, 25°C) was
irradiated with UV light and the kinetics of photochemical
reaction was monitored by UV-vis absorption measurements.
As shown in Figure 5a, the decrease in number of o-
notrobenzyl groups is reflected by the decreasing absorption
around 265 nm, while the increase of the amount of
nitrosobenzaldehye is indicated by its growing absorption
around 310 nm. This is in agreement with previous literature
reports.50 The transformation from hydrophobic o-nitribenzyl
ester to hydrophilic carboxyl derivative increased the
dissolution of conjugates.
Figure 5: (a) Irradiation time-dependent evolution of UV-vis absorption spectra recorded for the micellar dispersion of t-P56-b-NG5 (0.2 g/L, pH 7.4 PBS buffer,25°C). (b) Time-
dependent evolution of fluorescence emission spectra of NR-loaded t-P56-b-NG5 micellar dispersion (0.2 g/L, pH 7.4 PBS buffer,25°C) upon UV irradiation. (c) Time-dependent
evolution of fluorescence emission spectra of NR-loaded t-P56-b-BG5 micellar dispersion (0.2 g/L, pH 7.4 PBS buffer,25°C) upon UV irradiation. (d) Normalized emission intensities
recorded for NR-loaded t-P56-b-NG5 and NR-loaded t-P56-b-BG5 micellar dispersion upon UV irradiation.
6 | J. Name., 2012, 00, 1-3
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To further explore the disruption degree of hydrophobic
microstructural upon UV irradiation, we utilized Nile Red as a
polarity-sensitive fluorescence probe, as it is well-known that
Nile Red emission was quenched in water. Evolution of
fluorescence emission spectra (λex= 550 nm) of Nile Red loaded
t-P56-b-NG5 micelles (0.2 g/L, 25°C) in PBS buffer (pH 7.4) upon
UV irradiation was shown in Figure 5b. The fluorescence
intensity of Nile Red exhibited a progressive fluorescence drop
and then reached 14% of initial state after 30 min irradiation.
In contrast, no distinct changes were observed in emission
intensity for the Nile Red loaded without photolabile group t-
P56-b-BG5 micellar solution after 30 min UV irradiation (Figure
5c), indicating that the influence of photo bleaching was
sufficiently low. The true reason for the reduction of
fluorescence intensity was that photocleavage of o-nitribenzyl
ester linkages upon UV irradiation completely disrupted the
hydrophobic microstructural.
Figure 6: (a) Cumulative Dox release profile from drug-loaded hydrogel (pH 7.4 buffer,
37°C) without and with UV irradiation for 30 min. (b) Comparison of MTT cytotoxicity
assay results of blank and Dox-loaded micellad solution of t-P56-b-NG5 at
a
concentration of 3.0 g/L without irradiation (red bar) and with 365 nm UV irradiation
(blue bar) against HeLa cells. The data are expressed as mean ± sd (n = 3). *P < 0.05 (t-
test). Dox-loaded micelles induced apoptosis of HeLa cells as detected by DAPI nuclear
staining (405 nm excitation; 440-480 nm blue channel). CLSM images of HeLa cells after
incubation for 24 h in the presence of drug-loaded micelles without (c) and with (d) 365
nm UV irradiation for 30 min.
Photo-regulated Doxorubicin release profile and cytotoxicity of
Drug-loaded micelles
The Dox concentrations in the dialysate were quantified by
measuring the absorption intensities at 480 nm against
corresponding standard calibration curves (Figure S6), and
drug LC% was determined to be 4.3% and the LE% was
estimated to be approximately 45%. Figure 6a showed the
cumulative Dox release profiles from drug-loaded hydrogel
under varying conditions. At 37°C and in pH 7.4 buffer solution,
~10% of loaded drug can be released over 24 h for hydrogel
without UV irradiation. Whereas, the released rate of Dox
significantly accelerated after UV irradiation, caused ~80%
release of loaded drug in 24 h.
This conclusion was further proved via the DAPI nuclear stain
technique. Figure 6c showed CLSM images recorded for HeLa
cells after incubation for 24 h in the presence of Dox-loaded
micelles without (Figure 6c) and with (Figure 6d) 30 min of UV
irradiation. For cells treated with 30 min irradiation, the cell
viability was lower, which agreed with the MTT assay results
(Figure 6b).
Next, we examined the cytotoxicity of t-P56-b-NG5 micelles in
vitro cell via the MTT assay against HeLa cells (Figure 6b). The
cell viability of drug-free blank micelles with and without UV
irradiation remained to be ~85% at 3.0 g/L polymer
concentration, suggesting that PEG-polypeptide conjugates
micelles were almost noncytotoxic. When HeLa cells were
incubated with Dox-loaded micelles without UV irradiation,
~59% cells survived at 3.0 g/L polymer concentration. The
cytotoxicity results can be rationalized by highefficiency of
cellular uptake, ester hydrolysis and triggered drug release
under cellular enzymes and pH gradients environment. Dox-
loaded micelles after 30 min UV irradiation were added to cells
culture medium, the cell viability decreased further and only
39% cells survived. Indicating that Dox-loaded micelles
subjected to UV irradiation exhibited enhanced treatment
performance. The statistically significance difference could be
Conclusions
Here, we perpared a photo-degradable injectable self-healing
hydrogel based on star poly(ethylene glycol)-b-poly(γ-o-
nitrobenzyl-L-glutamate) by hydrophobic interactions. The
reversible hydrophobic interactions endowed hydrogels with
injectable and self-healing properties. Upon photo irradiation,
full cleavage of the o-nitrobenzyl ester was observed, resulting
in a complete hydrogel disruption and controlled release of
hydrophobic pharmaceutical. The increasing release ratio of
hydrophobic pharmaceutical Dox upon irradiation enhanced
apoptosis ratio of HeLa cell. Notably, the medical applications
of UV-responsive hydrogel are limited by the high energy of UV
radiation (which is harmful to human tissues) and their
insufficient tissue penetration, as well as their absorption by
biological component. Although UV light was applied for the
present study to trigger the gel-to-sol transition and drug
release, o-nitrobenzyl ester derivatives have been approved to
be responsive to near infrared (NIR) irradiation as well
possessing better tissue penetration.51-53 We expect that the
further integration of the current design with NIR cleavable
moieties will render the hydrogel with improved tissue
penetration and more appropriate for real clinical applications.
confirmed by Students test with p
＜0.05. This is also in
agreement with the discrepancy in drug release profiles for
nonirradiated and irradiated micellar dispersions (Figure 6a).
We further assessed the cytotoxicity of the nitrobenzyl alcohol
leaving group against HeLa cells (Figure S9), which showed
negligible toxicity toward cells.
Notes and references
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