From macro to micro to nano: The development of a novel lysine based hydrogel platform and enzyme triggered self-assembly of macro hydrogel into nanogel

Article abstract of DOI:10.1039/c4tb01902d

A novel L-lysine based multifunctional crosslinker family was developed and utilized to fabricate a 100% pure poly(ester amide) (PEA) hydrogel with unique structure via a novel gelation strategy in a single and rapid step. Enzyme triggered biodegradation

Full text of DOI:10.1039/c4tb01902d

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ARTICLE
From macro to micro to nano: development of a novel lysine based
hydrogel platform and the enzyme triggered self-assembly of macro
hydrogel into nanogel
Cite this: DOI: 10.1039/x0xx00000x
Received 00th OOXXXX,
Accepted 00th OO XXXX
a,c,
b
a,c
b,c*
DeꢀQun Wu Jun Wu , XiaoꢀHong Qin, and ChihꢀChang Chua,
DOI: 10.1039/x0xx00000x
A novel Lꢀlysine based multifunctional crosslinker family was developed and utilized to
fabricate 100% pure poly(ester amide) (PEA) hydrogel with unique structure via a novel
gelation strategy in a single and rapid step. Enzyme triggered biodegradation was utilized to
turn the resultant macro hydrogel into abundant microgels with a 3 micron size in the early
stage. Nanogels of 50 nm in diameter were then formed after 8 days biodegradation in enzyme
solution. The enzyme degradation of doxorubicin (DOX) loaded macroꢀgel indicated that the
micro and nano gels containing DOX could be obtained by the same strategy while keeping the
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sustained drug release performance. This study reported
a new biocompatible and
biodegradable crosslinker and hydrogel system and illustrated a new nanotechnology capable
of controllably producing nanogels by the enzymatic degradation of macro hydrogels.
1
. Introduction
hydrogel systems are very limited and they are all fabricated via
photocrosslinking with the assistance of polyethylene diacrylate
Biodegradable polymeric nanoparticles have been widely
or other polymers with diacrylate groups. This results in a very
slow biodegradation rate, and contributes to an uncontrollable
1
ꢀ4
utilized for drug delivery or theranostic applications. When
compared with nanoparticles, biodegradable nano size
polymeric hydrogels are more attractive for certain types of
drug delivery applications because of their high water content
and 3ꢀd porous network structure.
demonstrated by the macro size hydrogels.
preparing the nano size hydrogel in a reproducible and facile
24ꢀ26
and complicated biodegradation mechanism.
Herein, a new pure PEA hydrogel fabrication strategy was
developed, utilizing the theory of gelation in polymer
5
ꢀ10
This has been widely
27ꢀ29
chemistry.
For this purpose, multifunctional PEA
1
1ꢀ15
However,
monomers (f>2) are required in the polymerization to form gels
30
under certain conditions. However, to the best of our
1
6ꢀ19
manner is still challenging.
Here we proposed a novel, but
knowledge, there have not been any reported multifunctional
PEA monomers suitable for this goal. Therefore, a new Lꢀ
lysine based PEA monomer family was developed for the first
time in this report. Monomers Lysꢀx(x=4, 6, 8) were
synthesized according to a newly modified protocol by reacting
the Lꢀlysine with fatty diol under the existence of
toluenesulfonic acids. This water soluble monomer family has 4
functional primary amine groups (f=4) and is nontoxic to cells
even at high concentration. The 4ꢀarm functional monomer
family has shown robust crosslinking capability via its first
time application for fabricating hydrogels in this report.
simple and straightforward strategy to fabricate the nano size
hydrogels: biodegrade the macro size hydrogel into nano size
hydrogel, which will maximally keep the original unique
hydrogel property and structure.
To utilize this strategy, a specially designed macro hydrogel
platform is required with the following property/structure: (1)
high permeability and porosity allowing the enzyme to reach
the most interior sites of the hydrogel network; (2) selectively
distributed structures/sites for enzyme to attack and cut the
macrogel into desired shape and size; (3) suitable chemical
bonds for enzyme to attack. Based on the above requirements,
a lysine based poly(ester amide) (PEA) hydrogel platform
associated with a novel gelation strategy was developed for this
study. PEAs were selected here because they have shown
excellent
biodegradation behavior.
biocompatibility
and
controllable
enzyme
1
4, 20ꢀ23
However, the reported PEA
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Journal Na2Dme
reaction of Lysꢀ4 monomer and pꢀnitrophenyl diester
monomers at 80 C in DMAc. In briefly, NS (or NA, NSu) and
2
. Experimental
o
Lysꢀ4 monomers at a feed molar ratio 1.5 (or 2) were added to a
vial and dissolved in DMAc at 50 C, and then triethylamine
2
.1 Materials
o
(
TEA) (4.5 eq to Lysꢀ4) was added. The above mixed solution
Lꢀlysine monohydrochloride (Lys), pꢀtoluenesulfonic acid
monohydrate (TosOH·H2O), adipoyl chloride, sebacoyl
chloride, succinyl chloride, 1,4ꢀbutanediol, (Alfa Aesar, Ward
Hill, MA) and pꢀnitrophenol (J. T. Baker, Phillipsburg, NJ)
were used without further purification. Triethylamine (TEA)
from Fisher Scientific (Fairlawn, NJ) was dried via refluxing
with calcium hydride and then distilled. Other solvents, such as
o
was placed in an 80 C oil bath for specific time and macrogel
formation occurred, i.e., polymerization and crosslinking
reactions occurred simultaneously. The resultant macrogels
were removed carefully from the vials and washed with DMSO
and acetone several times and then with distilled water to
remove any residual chemicals. The distilled water was
replaced periodically. After this purification process, the
macrogels were soaked in distilled water until swelling
equilibrium and then removed and dried in vacuo at room
temperature for 48 hours for characterization.
benzene,
toluene,
ethyl
acetate,
acetone,
N,Nꢀ
dimethylacetamide (DMAc) and dimethyl sulfoxide (DMSO)
were purchased from VWR Scientific (West Chester, PA) and
were purified by standard methods before use. Trypsin (Type
IXꢀS, from bovine pancreas, lyophilized power, 13, 000ꢀ20,000
2
.5 Fourier transform infrared (FT-IR) spectroscope
BAEE
units/mg
protein),
hydralazine,
1ꢀ[3ꢀ
(
dimethylamino)propyl]ꢀ3ꢀethylcarbodiimide (EDC) and Nꢀ
FTꢀIR spectra of monomers and macro hydrogels were
recorded on a spectrophotometer (PerkinꢀElmer MagnaꢀIR560
Spectrometer) to characterize the chemical structures of Lysꢀ4
monomer and its hydrogels. The samples were ground into
powders, compressed onto the KBr crystal, and FTꢀIR spectra
were recorded in the wavenumbers range of 550ꢀ4,000 cmꢀ1.
FTꢀIR spectra were then obtained with a PerkinElmer (Madison,
WI) Nicolet Magana 560 FTIR spectrometer with Omnic
software for data acquisition and analysis.
Hydroxysuccinimide (NHS) were purchased from VWR
Scientific (West Chester, PA). 5ꢀ(and 6)ꢀCarboxyfluorescein
succinimidyl ester (NHSꢀfluorescein, excitation maximum 491
nm and emission maximum 518 nm) was purchased from
Pierce (Rockford, IL).
2
.2 Synthesis of the Lys-x monomers
As shown in Fig. 1, diꢀpꢀtoluenesulfonic acid salts of bisꢀLꢀ
lysine ester monomer (Lysꢀ4) was prepared as one example to
illustrate the Lysꢀx monomers library. Typically, Lꢀlysine
2
.6 Interior morphology of macrogels
(
0.132 mol), pꢀtoluenesulfonic acid monohydrate (0.132 mol),
Scanning electron microscopy (SEM) was employed to analyze
the interior morphological structure of the Lysꢀ4 macrogels as a
function of the precursor feed ratio before and after
biodegradation. Typically, after being incubated in distilled
water at room temperature for 3 days to reach its equilibrium
swelling, the macrogel was then gently removed and
immediately transferred into liquid nitrogen to freeze and retain
the swollen structure. The samples were subsequently freezeꢀ
dried for three days in a Virtis (Gardiner, NY) freeze drier in
and 1, 4ꢀbutanediol (0.06 mol) in 250 mL of toluene were
placed in a flask equipped with a DeanꢀStark apparatus, a
CaCl2 drying tube, and a magnetic stirrer. The solidꢀliquid
reaction mixture was heated to 90°C to reflux for 16 hours until
4
.3 mL (0.24 mol) of water was collected.
The reaction mixture was then cooled to room temperature,
filtered and dried, and finally purified by dissolving in
o
o
isopropyl alcohol at 40 C and cooling down at ꢀ20 C overnight
for precipitation of diꢀpꢀtoluenesulfonic acid salts of bisꢀLꢀ
lysine esters from isopropyl alcohol for three times. Isopropyl
alcohol was changed every time after precipitating, the white
o
vacuo at ꢀ50 C.
For the preparation of the particles generated from the
biodegraded macrogels for the SEM observation, a dropwise of
the solution medium where the Lysꢀbased PEA macrogel was
immersed was removed at preꢀdetermined periods of immersion
and placed onto a glass coverslip, dried in a hood overnight,
and finally fixed on aluminum stubs and coated with gold for
o
sticky mass was dried in vacuo at 40 C for 24 hours. The final
product was white powder, and the yields was 70~80%. This
new Lysꢀ4 monomer differs from prior published Lysꢀbased
monomers in two ways: no need of protection and deprotection
during the synthesis, and the availability of four amine groups
in each monomer instead of one functional carboxylic acid or
3
0 seconds for SEM observation with a Hitachi (Mountain
3
1, 32
View, CA) S4500 SEM instrument. Image analyses of SEM
data were performed by using the public domain NIH image
program.
amine group in the prior published studies.
2
.3 Synthesis of the monomers of di-p-nitrophenyl esters of
dicarboxylic acids
2
.7 Swelling kinetics of Lys-based PEA macrogel
The synthesis of the diꢀpꢀnitrophenyl succinate (NSu), diꢀpꢀ
nitrophenyl adipate (NA) and diꢀpꢀnitrophenyl sebacate(NS)
The swelling kinetics of the Lysꢀbased macrogels was
measured at room temperature (25 C). Dried Lysꢀbased
macrogel were weighed and immersed in 10 mL of distilled
water at room temperature for preꢀdetermined intervals; they
were then removed, and water on the macrogel surface was
gently wiped by wet filter paper and weighed until there was no
o
3
1
monomers was described in previous literatures
illustrated in Figure 1a.
and is
2
.4 Fabrication of Lysine based PEA polymers and their
macrogel
further weight change. The swelling ratio (
follows:
Q) was calculated as
The Lysꢀbased biodegradable and pure AAꢀPEA polymers and
their macrogels were prepared by directly polycondensation
2
| J. Mater. Chem. B, 2014
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DOI: 10.1039/C4TB01902D
Ws −Wd
Wd
solution containing nanogel, and the solution pH was adjusted
to 5.0. NHSꢀfluorescein was dissolved in DMSO and then
added to the above nanogel solution, the reaction lasted for 12
hours with stirring and the collected to be dialyzed against diꢀ
water using dialysis tube (MW cutoff 12,000) to remove the
unreacted NHSꢀfluorescein and solvent DMSO. Finally, the
resulting solution was free dried by the freeze drier to collect
the dyeꢀtagged Lysꢀbased PEA nanogel. To obtain the image of
the NHSꢀfluorescein attached nanogel, the freezeꢀdried nanogel
Q =
×100%
(1)
where Ws is the weight of the swollen macrogel at time t and
Wd is the weight of the dry macrogel at t = 0.
2
.8 Mechanical Testing
The mechanical testing of the Lysꢀbased macrogels was were dispersed in a PBS solution and sonicated for 10 min, then
performed on a DMA Q800 Dynamic Mechanical Analyzer observed by Fluorescent microscopy. Fluorescent microscopy
(
TA Instruments Inc., New Castle, DE) in a “controlled force” was performed using a confocal fluorescent microscope (C1ꢀsi,
mode. The swollen macrogels samples (disc of 1.2 cm diameter) Nikon, Japan) fitted with standard FITC and Texas Red filter
were submerged in distilled water and mounted between a sets.
movable compression probe (diameter 15 mm) and the fluid
cup. A compression force from 0.01 to 0.05 or 0.30 N 2.11 Nanogel size and zeta potential measurement and size
(
depending on the gel strength) at a rate of 0.02 or 0.05 N/min distribution
was applied at a room temperature. The compression elastic
modulus (E) of the swollen macrogel was calculated from the
plot the compressional stress versus strain.
The size, size distribution and zeta potential of the Lysꢀbased
PEA nanogel were determined by a Zeta sizer (Nano ZS,
Malvern Instruments). The nanogel contained aqueous solution
2
.9 In vitro enzymatic biodegradation of Lys-based PEA (200 mg/L) was passed through a 0.45
ꢀm pore size filter prior
macrogel
to measurement.
The biodegradation of Lysꢀbased macrogels was carried out in
a small vial containing a small piece of known weight dry
macrogels sample (ca. 50 mg) and 10 mL of PBS buffer (pH
2
.12 Drug Loading in Lys-based PEA nanogel and in vitro
Release
7
.4, 0.1 M) with trypsin at the concentration of 0.1 mg/mL. A
To investigate the DOX loading in the Lysꢀbased PEA nanogel,
Lysꢀ4 and NA monomers and DOX were dissolved in DMAc
together, and the DOXꢀimpregnated Lysꢀbased PEA hydrogels
were fabricated as described before as the fabrication of lysine
based PEA polymers and their macrogels. In a typical case, 10
mg DOX, 1.0 g Lysꢀ4 and 0.85 g NA were dissolved in DMAc
pure PBS buffer was used as a control. The vial was then
incubated at 37 C. The incubation media were refreshed daily
o
in order to maintain enzymatic activity. At predetermined
immersion durations, macrogels samples were removed from
the incubation medium, and then were washed with PBS three
times and then lyophilized in vacuo with FreeZone Benchtop
and Console Freeze Dry System (Model 7750000,
o
and heated to 80 C for 3 min to form DOXꢀimpregnated Lysꢀ
based PEA macrogel. The resultant macrogel was immersed in
acetone, DMSO and then diꢀwater to remove any unreacted
regents. The macrogel biodegradation procedure is the same as
described before in vitro enzymatic biodegradation of lysine
based macrogel, and the biodegradation time is 6 days in 0.1
mg/mL trypsin solution. The obtained Lysꢀbased PEA nanogel
containing DOX were dialyzed in a dialysis tube (MW cutoff at
o
LABCONCO Co., Kansas City, MO) at ꢀ48 C for 72 hours to a
constant weight. The degree of biodegradation was estimated
from the weight loss of the macrogels based on the following
equation:
W ꢀWt
0
W (%) =
l
(2)
W0
1
00,000 g/mol) for one day. After dialysis, the dialysis tube
was directly immersed into 400 mL PBS solution (pH=7.4,
.1M). Aliquots of 3 mL were subsequently withdrawn from
where Wl is the weight loss percent, W0 was the original
weight of the dried lysine based macrogel sample before
immersion, and Wt was the dried lysine based macrogel sample
weight after incubation for time t. The weight loss data from the
average of three specimens was recorded.
0
o
the PBS periodically at 37 C at predetermined time. After each
sampling, 3 mL fresh PBS solution was added back to the
dialysis tube to maintain a constant volume of the solution. The
amounts of DOX released from nanogel were measured by
To observe the nanogel generated from the macrogel using UV absorbance at 497 nm of the 3 mL aliquot removed at
biodegradation in the enzyme solution at predetermined time, predetermined periods (calibration curve of DOX not shown).
few drops of the biodegradation solution were collected and
placed on the silicon slides for SEM characterization.
To determine the drug entrapment efficiency and drug
loading efficiency of the Lysꢀbased nanogel, the amounts of
DOX released from the preloaded Lysꢀ4 based macrogel during
the swelling was tested (M1). Additionally, the amounts of
2
.10 Fluorescein Microscopy
The nanogels generated from the biodegraded Lysꢀbased DOX released in the course of biodegradation and dialysis of
hydrogels were modified by coupling a fluorescence dye for nanogel were calculated ( ). The DOX was measured by
M
2
visual purpose in subsequent biological studies. In brief, after using UVꢀVis absorbance at 497 nm and the amounts were
the macro hydrogel was biodegraded for 5 days in 0.1 mg/ml calculated according to the calibration curve. The final amounts
trypsin medium, the biodegradation solution was collected and of DOX loaded into the nanogel were calculated from:
dialyzed through the dialysis tube (MW cutoff 100,000) to
remove the trypsin enzyme. After dialysis, predetermined
amounts of NHSꢀfluorescein were added into the dialyzed
(3)
M =M −M −M
2
L
0
1
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Journal Na2Dme
where M0 is the initial amounts of DOX preloaded into the 3. Results and Discussion
macrogel during the macrogel formation.
The drug entrapment efficiency (DEE) and drug loading 3.1 Synthesis of the Lys-x monomers and hydrogels
efficiency(DLE) in the biodegradation nanogel are defined as
follows.
Polymerization occurred between lysine monomers and diacid
based monomers (NA, NS) (Fig. 1a and 1b) via one single step
polycondensation reaction (Fig. 1c). The H NMR of lysine
Mass of loaded drug in nanogel
(
4)
DEE =
×100%
×100%
1
Mass of drug fed initially
monomer was illustrated in Fig. 2a, the chemical shift δ at 8.34
Massof loadeddrugin nanogel
Massof drug loadednanogel
(
5)
ppm is attributed to amine group attached onꢀNH TosOH (d), δ
DLE =
2
at 8.02 ppm (hydrogen chloride attached to amine group, l), δ at
7
.75, 7.14 ppm (phenyl group of TosOH attached to amine
2
.13 In vitro cytotoxicity of Lys-based PEA nanogels
00 µL of Hela cells in DMEM medium with a cell
group, b, c.), δ at 4.15 ppm(e, ꢀC=OꢀOꢀCH ) δ at 3.81 ppm (g, ꢀ
C=OꢀOꢀCHꢀNH ), δ at 2.75 ppm (k, ꢀCH ꢀNH ), δ at 2.21ppm
2
2
2
2
2
(
a, phenylꢀCH ), δ at 1.78 ppm (j, ꢀCH ꢀCH2ꢀCH ꢀ), δ at 1.68
4
3
2
2
concentration of 1.00 ×10 cells/mL are added to each well in a
9
After incubation for 24 h in an incubator (37 C, 5% CO ), the
3
ppm (h,ꢀCHꢀCH
at 1.51 ppm (i, ꢀCH ꢀCH2ꢀCH ꢀ). FTꢀIR: 3640 cm (ꢀNH2,
2 2
ꢀCH
2
ꢀ), δ at 1.59 ppm (f, ꢀCH
2
2
ꢀCH2ꢀCH ꢀ) δ
2
6ꢀwell plate. The number of Hela cells in each well is 2.0×10 .
ꢀ
1
o
2
ꢀ
1
culture medium was replaced by 200 µL of DMEM containing wide), 1745 cm (ꢀC=OꢀOꢀ). The final products after
the FITCꢀlabeled 4ꢀLysꢀ4 nanogel [4ꢀLysꢀ4(1.5:1)ꢀFITC] or polymerization could be polymers or crosslinked polymers
DOXꢀloaded 4ꢀLysꢀ4(1.5:1)ꢀFITC nanogel at predetermined
nanogel concentrations, and the mixture was further incubated
for another 48 hours. Then, the DMEM medium containing
either 4ꢀLysꢀ4(1.5:1)ꢀFITC nanogel or DOXꢀloaded 4ꢀLysꢀ
(
1
macro gels), depending mainly on the monomer ratios (Fig.
(c)). The formation of the hydrogels was demonstrated by FTꢀ
ꢀ1
IR in Fig. 2c, the characteristic bands of 1742 cm attribute to
C=O stretch; 1640 cm attribute to amide I; 1560 cm attribute
to amide II. These corresponding characteristic bands at 1742
ꢀ
1
ꢀ1
4
(1.5:1)ꢀFITC nanogel were replaced by fresh DMEM and 20
µL of MTT solution (5 mg/mL) was added to the Hela cells in
ꢀ
1
ꢀ1
ꢀ1
each 96ꢀwell plate. After incubation for 4 h, 200 µL of DMSO cm , 1640 cm and 1560 cm were observed in 4ꢀLysꢀ4(1.5:1),
was added and shaken at room temperature. The optical density 4ꢀLysꢀ4(2:1), 8ꢀLysꢀ4(1.5:1) and 8ꢀLysꢀ4 (2:1) hydrogels. It
(
OD) was measured at 570 nm with a Microplate Reader
can be verified that hydrogels were formed via the
condensation reaction of amino groups in Lysꢀ4 and carbonyl
groups of NA or NS. As demonstrated in Figure 2b, the
Model550 (BIOꢀRAD, USA). The cell viability was calculated
by the following equation. Cell viability= ODtreated/ODcontrol,
where ODcontrol was obtained in the absence of nanogels
ꢀ
1
characteristic bands of 3300 cm attribute to NꢀH stretch, the
(
DMEM medium only) and ODtreated was obtained in the
presence of nanoparticles with or without DOX. All cell corresponding strength of 4ꢀLysꢀ4 (1.5:1) is bigger than that of
viability data are normalized against the blank controls (no the 4ꢀLysꢀ4 (2:1) due to the different feed ratio NA to Lysꢀ4.
nanogels and no DOX).
As for the former, the feed ratio NA to Lysꢀ4 is 1.5 to 1, that
means there are more free amino groups in the hydrogels, the
2
.14 Cell internalization of 4-Lys-4-FITC biodegradation
same case was observed in 8ꢀLysꢀ4 (1.5:1) and 8ꢀLysꢀ4 (2:1)
hydrogels. Below the gelling point ratio, soluble polymers will
be formed and above the point, the gel will form. After
optimization, it was found that the gel point is mainly affected
nanogel
Hela cells were used for the cell internalization study of FITCꢀ
tagged Lysꢀbased PEA [4ꢀLysꢀ4(1.5:1)] nanogel with or
without DOX [4ꢀLysꢀ4(1.5:1)ꢀFITC]. Hela cells were cultured by: reaction temperature, the monomer ratio, and concentration
on LabTek chamber slide dishes (2.0×10 cells/plate) to grow to of the monomer (Table 1). Compared to the reported PEA
4
about 70% confluence. Culture medium was then removed, and
mL of fresh DMEM containing 4ꢀLysꢀ4(1.5:1)ꢀFITC nanogel
1 mg/mL) with or without DOX was added into each well. All
hydrogel systems, this new Lysine strategy has a few unique
advantages, including: 1) one step hydrogel fabrication by
simply mixing and heating the monomer solutions together
within minutes, 2) eliminating the reported need of the tedious
protection and deprotection procedures of the amino acids that
1
(
the cells were incubated in the plates containing DMEM and
o
polymers for 6 h, 12 h, 24 h at 37 C, respectively. Then the
medium was removed and cells were washed with PBS solution
three times. Then, 1 mL of PBS solution was added into each would be costly, time consuming and result in lower yields, 3)
3
1
well. The fluorescent images of the cells were observed under
excitation at 488 nm using confocal laser scanning microscopy
double bonds free gelation, 4) hydrogel having 100% pure PEA
component. Subsequently, a 100% pure lysine based PEA
hydrogel library was developed here and the detailed gel
synthesis protocol could be found in the supporting information.
Here is one simple example for 4ꢀLysꢀ4 (xꢀLysꢀy, where x and
y represent the methylene in the diacid monomer and diol
monomer, respectively.) gel: 4ꢀLysꢀ4 gel was prepared by
mixing NA and Lysꢀ4 with the molar ratio 2:1 for 2.0 min at 80
(
CLSM) (Leica TCS SP2AOBS, Germany).
o
C.
4
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DOI: 10.1039/C4TB01902D
Table 1. Preparation of Lysꢀ4 based macrogels.
sample NA
code
NS
Lysꢀ Lysꢀ Lysꢀ DMAc Et N
3
4
6
8
4
4
4
4
8
4
8
4
4
6
4
6
4
8
4
8
8
6
8
6
8
8
8
8
ꢀLysꢀ 0.76g
(1.5:)
ꢀLysꢀ 1.02g
(2:1)
\
\
1.0g
\
\
2.0 g
2.0 g
2.0 g
2.0 g
2.0 g
2.0 g
0.65g
0.65g
0.65g
0.65g
0.65g
0.65g
0.65g
0.65g
0.65g
1.0g
\
\
ꢀLysꢀ
(1.5:)
ꢀLysꢀ
(2:1)
\
0.87g 1.0g
1.16g 1.0g
\
\
\
\
\
\
\
ꢀLysꢀ 0.67g
(1.5:)
ꢀLysꢀ 0.95g
(2:1)
ꢀLysꢀ 0.65g
(1.5:)
ꢀLysꢀ 0.89g
(2:1)
\
\
\
\
\
\
\
\
\
\
1.0g
1.0g
\
\
1.0g 2.0 g
1.0g 2.0 g
\
\
Fig. 1 (a) Chemical structure of the diꢀpꢀnitrophenyl monomers. (b)
Chemical structure of the Lysꢀx monomer. (c) Illustration of
chemical structure of hydrogel. The Lysꢀx part in the structure
ꢀLysꢀ
(1.5:)
ꢀLysꢀ
(2:1)
ꢀLysꢀ
(1.5:)
ꢀLysꢀ
(2:1)
\
\
\
\
0.81g
1.08g
0.76g
1.01g
1.0g
\
\
2.0 g
2.0 g
1.0g
0.65g indicates the monomer shown in (b), pꢀToluenesulfonic acid and
hydrochloride salt of Lysine diester. The wave lines represent the
AAꢀPEA chains.
\
\
1.0g 2.0 g
1.0g 2.0 g
0.65g
0.65g
The polymerization/gelation rate of Lysꢀ4 with NS is faster
o
than with NA in DMAc at 80 C, e.g., 2 min vs. 4 min. The
polymerization/gelation of Lysꢀ4 monomers with Nsu monomer
(
x = 1 in Fig. 2b) under the same reaction condition as with NA
or NS, however, was unsuccessful. This was due to the less
methylene or activity in the Nsu monomer that formed short
spacer when polycondensation occurred, which made it difficult
to fabricate hydrogel.
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J. Mater. Chem. B, 2014 | 5

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Table 2 Compressive modulus of Lysꢀ4 based macrogels
Sample code
Monomer
feed ratio
molar Compressive
modulus (KPa)
4
4
8
8
ꢀLysꢀ4(1.5:1)
ꢀLysꢀ4(2:1)
ꢀLysꢀ4(1.5:1)
ꢀLysꢀ4(2:1)
NA: Lysꢀ4=1.5:1
NA: Lysꢀ4=2:1
NS: Lysꢀ4=1.5:1
NS: Lysꢀ4=2:1
25±4.5
46±6.1
670±53
1350±126
To successfully obtain nanogels via an enzyme biodegradation
strategy, one of the key factors is the three dimensional
structure of macrogel, which is directly determined or related to
the intrinsic chemical structure of the macro hydrogel. Before
the biodegradation study, the interior morphology of the freeze
dried Lysꢀx(x=4, 6, 8) based macro hydrogel library was
systematically investigated by scanning electron microscopy
(
SEM). As shown in Fig. 2e, before biodegradation, all the
hydrogels exhibited wellꢀdefined, threeꢀdimensional porous and
completely connected network structures with an average wall
thickness around 1.0ꢀ4.0 µm and average pore size ranging
from 4.0 to 10.0 ꢁm. It was found that x, y and m to n ratio will
affect the pore size and wall thickness. For example, as shown
in Fig. 2e, the average pore sizes of the hydrogel are 6.0 ꢁm
and 4.0 ꢁm for 4ꢀLysꢀ4 (1.5:1) (1.5:1 is molar ratio of monomer
NA to Lysꢀ4) and 4ꢀLysꢀ4 (2:1), and 10.0 ꢁm and 8.0 ꢁm for 8ꢀ
Lysꢀ4 (1.5:1) and 8ꢀLysꢀ4 (2:1). The 4ꢀLysꢀ4 hydrogels had
thinner cell walls than the 8ꢀLysꢀ4 hydrogels, which means 8ꢀ
Lysꢀx hydrogels had a more compact structure and the average
pore size decreased with the increasing of x. With the
increasing of amide group density formed in the hydrogel, i.e.,
the increasing crossꢀlinking density, the average pore size
decreased, which is consistent with the swelling ratio and
mechanics data of the macro hydrogels (Fig. 2b).
1
Fig. 2 (a) H NMR spectrum of Lysꢀ4 monomer (300 MHz, DMSOꢀ
d ). (b) Swelling kinetics of Lysꢀ4 based macrogels in distilled water
at 25 C. (c) FTꢀIR of lysine based hydrogel. A: 4ꢀLysꢀ4(2:1); B: 4ꢀ
Lysꢀ4(1.5:1); C: 8ꢀLysꢀ4(2:1); D: 8ꢀLysꢀ4(1.5:1). 1742 cm attribute
to C=O stretch; 1640 cmꢀ1 attribute to amide I; 1560 cm attribute
to amide II. (d) Optical images of hydrogels. A: 8ꢀLysꢀ4 (1.5:1); B:
ꢀLysꢀ4 (1.5:1). The molar ratio of 1.5:1 indicates NA or NS to Lysꢀ
monomer. (e) SEM images of Lysꢀ4 based hydrogels before
degradation. A: 8ꢀLysꢀ4(2:1); B: 8ꢀLysꢀ4(1.5:1); C: 4ꢀLysꢀ4(2:1); D:
6
o
ꢀ
1
ꢀ
1
4
4
4
ꢀLysꢀ4(1.5:1).
3
.2 Swelling kinetics of Lys-based PEA macrogel and
3
.3 In vitro enzymatic biodegradation of Lys-based PEA
mechanical properties
macrogel
Fig. 1c is the illustration of hydrogel crosslinking by the
reaction of the monomer A and monomer B to form hydrogel.
The enzyme biodegradation mechanism of the crossꢀlinked
lysine based hydrogels was subsequently investigated by a
Interestingly, it was found that the polymerization/gelation rate hydrolytic enzyme with a series of concentrations in PBS buffer
and gel structure are determined by the monomer structure solutions. Weight loss, size distribution and SEM were used to
characterize and monitor the biodegradation progress of the
under fixed reaction condition. For example, Lysꢀ4 shows a
hydrogels. The hydrolytic enzyme trypsin is utilized here as a
faster gelation rate when reacting with NS than with NA in
model enzyme because of its general degradation capability.
o
DMAc at 80 C, e.g., 2.0 min vs. 4.0 min. The
And we are focusing on the material related properties in this
polymerization/gelation of Lysꢀ4 monomers with Nsu monomer
(
study. The results indicated that the degradation behaviors are
x = 1 in Figure 1b) under the same reaction condition, however, affected by a variety of factors, including crosslinking density,
was only able to get very soft hydrogel or viscous mixture. This concentration of enzyme, biodegradation time, the density of
the hydrophobic moiety, and the molecular weight of the
backbone chain. For example, as shown in Fig. 4a, the
degradation behavior of lysine based hydrogel depended on
crosslinking density of amide groups and the hydrophobicity of
the hydrogels. For the 4ꢀLysꢀ4 (1.5:1) hydrogel immersed in
PBS solution without trypsin, its weight loss was only 5.0 wt%
may be due to the weaker reactivity or less methylene of the
Nsu monomer, which made it difficult to fabricate hydrogel.
The modulus of the resultant hydrogels was also affected by the
monomer structure: higher x or y would cause higher modulus
(
Table 2). For example, 8ꢀLysꢀ4 hydrogel had significantly
higher compression modulus (more than 25 times) than 4ꢀLysꢀ4 after 10 days, which means the factor from the hydrolytic
degradation of the buffer is very small or negligible. Therefore,
the degradation contribution is mainly due to enzyme attacking.
The biodegradation rate of hydrogel could be regulated by
tuning the crosslinking density. For example, 4ꢀLysꢀ4(1.5:1) is
faster than 4ꢀLysꢀ4(2:1), which is ascribed to the lower
crosslinking density of the amide groups in the hydrogels. With
the ratio of the monomer NA to Lysꢀ4 increased, the hydrogel
has the higher crosslinking density of the amide groups. A
hydrogel at the same monomers’ feed ratio: 670 ± 53 KPa vs.
2
5 ± 4.5 KPa (Table 2).
6
| J. Mater. Chem. B, 2014
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similar trend was observed in 8ꢀLysꢀ4 hydrogels. In general, the biodegradation is incomplete (Fig. 3b). Meanwhile, the average pore
degradation rate of hydrogel is related to several network size of the hydrogels increased during the biodegradation. As shown
parameters, such as the number of crosslinkers per backbone in Fig. 3c, the average pore size of the 8ꢀLysꢀ4 (1.5:1) and 8ꢀLysꢀ4
chain, molecular weight of the backbone unit, and the (2:1) was around 10.0 ꢁm and 6.0 ꢁm respectively, and the size for
proportion of biodegradable groups in the main and side 4ꢀLysꢀ4 (1.5:1) and 4ꢀLysꢀ4 (2:1) was around 12.0 ꢁm and 10.0 ꢁm
33ꢀ35
chain.
Thus, the biodegradation rate decreased with respectively. After 4 days, the average pore size of the 8ꢀLysꢀ4
increasing crosslinking density. It was found that the hydrogel (1.5:1), 8ꢀLysꢀ4 (2:1) was around 20.0 ꢁm and 15.0 ꢁm respectively;
with a higher swelling ratio had a faster biodegradation rate in the average size for 4ꢀLysꢀ4 (1.5:1), 4ꢀLysꢀ4 (2:1) was 30.0 ꢁm and
our study，
which is consistent with the above rules. After 40.0 ꢁm respectively, and the regular 3ꢀD structure of the
screening, 8ꢀLysꢀ4 and 4ꢀLysꢀ4 gels were chosen for the hydrogels was almost broken (Figures 3d). The Zeta potential
following study.
of 4ꢀLys4 (1.5:1) and 4ꢀLysꢀ4(2:1) nanogel was 14.5 mv and
.6 mv respectively, indicating net free primary amine groups
9
on the microsphere surface.
At 5 days, the microspheres on the hydrogel surface were
partially biodegraded and some of the broken microsphere was
observed (Fig. 3d). The hydrogel network almost collapsed and
the average pore size enlarged. We also investigated the status
of the biodegradation solution during the enzyme
biodegradation treatment. On the sixth day, a large number of
microgels were obtained by degradation, and the size decreased
to about 200 nm (Fig. 4b). After 8 days’ biodegradation, the
size of obtained nanogels was about 50 nm with a narrow size
distribution (Fig. 4c). The sizes of micro and nano gels in PBS
solution were characterized by DLS. The enzyme trypsin was
removed by dialysis tube (cutoff weight 100,000).
Fig. 3 (a) SEM images of the Lysine based macrogels with the
enzyme trypsin concentration of 0.1 mg/mL in PBS buffer (pH
7
.4, 0.1 M) after 1 day biodegradation. A: 4ꢀLysꢀ4(1.5:1); B: 4ꢀ
Lysꢀ4(2:1); C: 8ꢀLysꢀ4(1.5:1); D: 8ꢀLysꢀ4(2:1). (b) SEM
images of the lysine based hydrogel 4ꢀLysꢀ4(1.5:1) with the
enzyme trypsin concentration of 0.1 mg/mL in PBS buffer (pH
7
.4, 0.1 M) after 3 days biodegradation. (c) SEM images of the
lysine based macrogels with the enzyme trypsin concentration
of 0.1 mg/mL in PBS buffer (pH 7.4, 0.1 M) after 4 days
biodegradation. A: 4ꢀLysꢀ4 (1.5:1); B: 4ꢀLysꢀ4 (2:1); C: 8ꢀLysꢀ
4
(1.5:1); D: 8ꢀLysꢀ4 (2:1). Microgels and nanogels are
invisible because most of they have been washed out by PBS
for three times. (d) SEM images of the lysine based macrogels
with the enzyme trypsin concentration of 0.1 mg/mL in PBS
buffer (pH 7.4, 0.1 M) after 5 days biodegradation. A: 4ꢀLysꢀ
Fig. 4 (a) The weight loss of the lysine based hydrogels during
degradation. The hydrogels were immersed in PBS buffer (pH
7
4
(1.5:1); B: 4ꢀLysꢀ4(2:1); C: 8ꢀLysꢀ4(1.5:1); D: 8ꢀLysꢀ4(2:1).
.4, 0.1 M) with trypsin at the concentration of 0.1 mg/mL and
pure PBS for control. (b) SEM images of the Lysine based A:
ꢀLysꢀ4(1.5:1) macrogels with the enzyme trypsin
concentration of 0.1 mg/mL in PBS buffer (pH 7.4, 0.1 M) after
SEM was used to monitor the morphology change of the swollen A: 6 days biodegradation; B: 7 days biodegradation; C: DLS of
ꢀLyꢀ4 and 4ꢀLysꢀ4 hydrogels during enzymatic biodegradation. On
days biodegradation nanogel; D: DLS of days
Microgels and nanogels are invisible because most of they have
been washed out by PBS for three times.
4
8
6
7
the second day of biodegradation, there are a lot of regular biodegradation nanogel. (c) SEM images of the lysine based A:
microspheres on the hydrogel surface and in the hydrogels networks, 4ꢀLysꢀ4(1.5:1) hydrogels with the enzyme trypsin concentration
and the average size is ranging from 0.5 ꢁm to 4.0 ꢁm (Fig. 3a). This of 0.1 mg/mL in PBS buffer (pH 7.4, 0.1 M) after A: 8 days
is the first time that synthesized hydrogel was reported to be able to biodegradation; B: 9 days biodegradation; C: DLS of 8 days
produce microspheres (microgels) after being treated with enzyme. biodegradation nanogels; D: DLS of 9 days biodegradation
With the prolonged biodegradation time, the enzymatic nanogels.
biodegradation of hydrogel network continues and the average size
of the microsphere decreased to below 3.0 ꢁm after 2 days. The
morphology of the microspheres became less regular and some of
st
the microspheres linked like a string of beads, indicating the 1 stage
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3
.4 Drug Loading in Lys-based PEA nanogel and in vitro 3.5 Cell internalization of 4-Lys-4-FITC biodegradation
Release nanogel
The biodegradation behavior of the drug loaded Lys based Monomer Lysꢀ4 has 4 free amine groups, when the molar ratio
macro hydrogels were further examined using the DOX as a of NA to lysine based momomer Lysꢀ4 is 1.5:1, the resultant 4ꢀ
model drug. The hydrogels were biodegraded using 0.1 mg/mL Lysꢀ4 (1.5:1) hydrogel has free amine groups after hydrogel
trypsin PBS solution as described in the previous section. The formation compard to 4ꢀLysꢀ4 (2:1) hydrogel. The free amine
DOX was preloaded into the hydrogels during the macro groups endow the nanogel positive charge for in vitro cellular
hydrogel fabrication. In this study, for a 5.0 wt% drug added, uptake and chemical conjugation, such as fluorescence dye
the drug entrapment efficiency (DEE) and drug loading attachment. CLSM was used to investigate 4ꢀLysꢀ4(1.5:1)ꢀ
efficiency (DLE) was 51.6% and 2.6 %. The DOX loaded FITC biodegradation nanogels and DOX loaded 4ꢀLysꢀ
nanogels were obtained and chosen for release study after 5 4(1.5:1)ꢀFITC nanogel’s uptake by Hela cells and subsequent
days’ degradation. The obtained resultant nanogels were accumulation. Fluoresence dye, FITC, was chemically
washed with PBS before testing the DOX release. The yield of conjugated to the nanogel by reacting with its amine groups and
the above 4ꢀLysꢀ4 (1.5:1) nanogel was 18.6%. As demonstrated was used to track the nanogel in the cell for imaging. After 6 h
in Figure 5b, at time of 5 hours, the cumulative DOX release incubation with DOX loaded 4ꢀLysꢀ4(1.5:1)ꢀFITC nanogels
o
from the nanogels in both PBS solution (pH 7.4, 0.1M) and suspension (1 mg/mL) at 37 C, fluorescence was observed in
enzyme trypsin solution (0.1 mg/ml) was about 10.0%. Hela cells (Fig. 6). With the incubation time increasing, higher
However, release of DOX became a virtually constant first fluorescence intensity was observed in Figure 6B2. When the
order rate after 15 hours over a 10ꢀday period for nanogels in Hela cell grew to 24 h with the DOX loaded 4ꢀLysꢀ4(1.5:1)ꢀ
PBS solution (pH 7.4, 0.1M, 0.1 mg/ml trypsin).
FITC nanogels, a much higher intensity of the red fluorescence
was seen inside the Hela cells. This is consistent with the MTT
result of the DOX loaded 4ꢀLysꢀ4(1.5:1)ꢀFITC nanogels in
Figure 5c. The cytotoxicity increased with the increased
concentration of DOX loaded 4ꢀLysꢀ4(1.5:1)ꢀFITC nanogels.
When the concentration of DOX loaded 4ꢀLysꢀ4(1.5:1)ꢀFITC
nanogels is 0.25 mg/mL, the cell viability is less than 20%.
When the concentration is 0.5 mg/mL, the cell viability
decreased to 10%. Finally, at the concentration of 1.0 mg/mL,
the cell viability decreased to 8%.
With the degradation of the ester and amide bonds of the
hydrogel, the hydrophilic lysine segments of the nanogel
dissolved and diffused into the buffer solution, and nanogel
became more and more loose. When the degradation rate of the
lysine based nanogel came to a moderate stage, the released
DOX amounts increased in a linear relationship as a function of
the time (1st order). After degrading the 4ꢀLysꢀ4 (1.5:1)
nanogel, the release behavior came to an end. This indicates
that the release of DOX was controlled mainly by the
dissolution and dissociation of the 4ꢀLysꢀ4 (1.5:1) nanogels,
rather than by a diffusion mechanism.
Fig. 5 (a) CLSM images of the biodegradation particles: (A)
Em 595 nm for DOX; (B) em 515 nm for FITC. (C) Physically
overlaid image of panels A and B. (b) Cumulative release of
DOX from 4ꢀLysꢀ4 (1.5:1) hydrogel
biodegraded nanogels
at 37 °C in PBS solution (pH 7.4, 0.1 M) and the concentration
of enzyme trypsin is 0.1 mg/mL. (c) Cytotoxicity of the 4ꢀLysꢀ
Fig. 6 Confocal laser scanning microscopy images of Hela cells
for 6 h (A1, B1, C1,D1) 12 h (A2, B2, C2,D2), 24 h (A3, B3,
C3,D3) and free DOX (A0, C0, D0) incubating with DOX
loaded 4ꢀLysꢀ4(1.5:1)ꢀFITC nanogels and free DOX (20
4
(1.5:1)ꢀFITC nanogels, DOX loaded 4ꢀLysꢀ4(1.5:1)ꢀFITC
nanogels and free DOX at different concentrations. Control for
free DOX indicates no DOX for cell culture.
o
µg/mL) as control in DMEM medium at 37 C. (A1, A2, A3,
A0: bright field images, B1, B2, B3, B4: confocal green
fluorescence images; C1, C2, C3, C4: confocal red fluorescence
images; D1, D2, D3, D4: Merge.
8
| J. Mater. Chem. B, 2014
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DOI: 10.1039/C4TB01902D
9
Z. Gu, M. Yan, B. Hu, K. I. Joo, A. Biswas, Y. Huang, Y. Lu, P.
Wang and Y. Tang, Nano Letters, 2009, 9, 4533ꢀ4538.
4. Conclusions
In conclusion, a novel strategy is utilized to develop the 10 Q. Hu, P. S. Katti and Z. Gu, Nanoscale, 2014, 6, 12273ꢀ12286.
functional microgels and nanogels from the macrogels through 11 D. Ma and L.ꢀM. Zhang, Biomacromolecules, 2011, 12, 3124ꢀ
the controlled enzyme biodegradation of the screened
macrogels. To achieve the goal of nanogels fabrication, a new
multifunctional Lꢀlysine based diester monomer was specially
developed and a corresponding crosslinking method was used
to chemically fabricate a new family of pure amino acidꢀbased
poly(ester amide) hydrogels with desired chemical structures.
3
130.
1
1
2 M. Molinos, V. Carvalho, D. M. Silva and F. M. Gama,
Biomacromolecules, 2012, 13, 517ꢀ527.
3 D. Q. Wu, Y. X. Sun, X.ꢀD. Xu, S. X. Cheng, X. Z. Zhang and
R. X. Zhuo, Biomacromolecules, 2008, 9, 1155ꢀ1162.
For some screened macrogels, it was found that spherical 14 D. Q. Wu, J. Wu and C.C. Chu, Soft Matter, 2013, 9, 3965ꢀ3975.
nanogels with controllable size were formed and the final size 15 A. GuiseppiꢀElie, S. I. Brahim and D. Narinesingh, Adv. Mater.,
could be below 50 nm after being biodegraded for 8 days. With
2
002, 14, 743ꢀ+.
the abundance of free amine groups, the degraded microgel and
nanogels could easily conjugate with biological active reagents
or dyes and demonstrate a controllable and sustained release,
thus expand the biomedical applications of this gel platform.
1
1
6 M. Ahmed and R. Narain, Mol. Pharmaceut., 2012, 9, 3160ꢀ3170.
7 A. Concheiro and C. AlvarezꢀLorenzo, Adv. Drug Deliver.
Rev., 2013, 65, 1188ꢀ1203.
8 J. H. Ryu, R. T. Chacko, S. Jiwpanich, S. Bickerton, R. P. Babu
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Acknowledgements
1
7235.
This research is supported by a grant from the Rebecca Q. 19 M. H. Xiong, Y. Bao, X. Z. Yang, Y. C. Wang, B. Sun and J.
Morgan Foundation. The research is also supported by the
Fundamental Research Funds for the Central Universities, and
sponsored by Shanghai Pujiang Program 14PJ1400300. The
authors are grateful for Nanobiotechnology Center (NBTC) for
using research facilities in Cornell University for cell related
work.
Wang, J. Am. Chem. Soc, 2012, 134,4355ꢀ4362.
0 J. Wu and C. C. Chu, Acta. Biomater., 2012, 8, 4314ꢀ4323.
2
2
1 J. Wu, D. Wu, M. A. Mutschler and C.C. Chu, Adv. Funct. Mater.
012, 22, 3815ꢀ3823.
2 J. Wu and C. C. Chu, J. Mater. Chem. B, 2013, 1, 353ꢀ360.
3 J. Wu, D. Yamanouchi, B. Liu and C. C. Chu, J. Mater. Chem.
012, 22, 18983ꢀ18991.
,
2
2
2
,
Notes
2
a
Key Laboratory of Textile Science & Technology, Ministry
2
2
4 L. Almany and D. Seliktar, Biomaterials, 2005, 26, 2467ꢀ2477.
5 M. GonenꢀWadmany, R. Goldshmid and D. Seliktar,
Biomaterials, 2011, 32, 6025ꢀ6033.
6 X. Pang and C. C. Chu, Polymer, 2010, 51, 4200ꢀ4210.
7 S. Kirchhof, F. P. Brandl, N. Hammer and A. M. Goepferich,
J. Mater. Chem. B, 2013, 1, 4855ꢀ4864.
Education; College of Textiles, Donghua University, No.2999
North Renmin Road, Songjiang, Shanghai, 201620, China.
b
Department of Biomedical Engineering, Cornell University,
2
2
Ithaca, NY, 14853, USA.
c
Department of Fiber Science and Apparel Design, Cornell
University, Ithaca, NY, 14853, USA.
2
2
8 R. J. Sheridan and C. N. Bowman, Macromolecules, 2012, 45,
7
634ꢀ7641.
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Graphic table
Development of a novel lysine based hydrogel platform and the enzyme triggered self-
assembly of macro hydrogel into nanogel