Volume 3 Number 11 21 March 2015 Pages 22332412

Journal of

Materials Chemistry B
Materials for biology and medicine
www.rsc.org/MaterialsB

ISSN 2050-750X

PAPER
Xiao-Hong Qin, Chih-Chang Chu et al.
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

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PAPER

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2286

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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
De-Qun Wu,ac Jun Wu,b Xiao-Hong Qin*ac and Chih-Chang Chu*bc
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 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 of
biodegradation in the enzyme solution. The enzyme degradation of doxorubicin (DOX) loaded macro-

Received 17th November 2014

Accepted 8th January 2015

gel indicated that the micro and nano gels containing DOX could be obtained using the same strategy
while retaining their sustained drug release performance. This study reports a new biocompatible and

DOI: 10.1039/c4tb01902d

biodegradable crosslinker and hydrogel system, and illustrates a new nanotechnology capable of

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controllably producing nanogels using the enzymatic degradation of macro hydrogels.

1. Introduction
Biodegradable polymeric nanoparticles have been widely
utilized for drug delivery or theranostic applications.14 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 3D porous network structure.510 This has been widely demonstrated by macro size hydrogels.1115 However, preparing a nano
size hydrogel in a reproducible and facile manner is still challenging.1619 Herein, we propose a novel, but simple and
straightforward strategy to fabricate nano size hydrogels: the
biodegradation of macro size hydrogel into nano size hydrogel,
which will maximally retain the original unique hydrogel
property and structure.
To utilize this strategy, a specially designed macro hydrogel
platform is required with the following properties/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 the enzyme to attack and cut the
macrogel into the desired shape and size; and (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 been
shown to exhibit excellent biocompatibility and controllable
enzyme biodegradation behavior.14,2023 However, the reported
PEA hydrogel systems are very limited and they are all fabricated
via photocrosslinking with the assistance of polyethylene diacrylate or other polymers containing diacrylate groups. This
results in a very slow biodegradation rate and contributes to an
uncontrollable and complicated biodegradation mechanism.2426
Herein, a new pure PEA hydrogel fabrication strategy
was developed, utilizing the theory of gelation in polymer chemistry.2729 For this purpose, multifunctional PEA monomers (f > 2)
are required in the polymerization to form gels under certain
conditions.30 However, to the best of our knowledge, there have
not been any reports on multifunctional PEA monomers suitable
for this goal. Therefore, a new L-lysine based PEA monomer family
was developed for the rst time in this report. Monomers Lys-x (x
4, 6, 8) were synthesized according to a newly modied protocol
by reacting L-lysine with a fatty diol in the presence of toluenesulfonic acid. This water soluble monomer family has 4 functional primary amine groups (f 4) and is non-toxic to cells even
at high concentrations. The 4-arm functional monomer family
has shown robust crosslinking capability via its rst time application for fabricating hydrogels in this report.

Key Laboratory of Textile Science & Technology Ministry of Education, College of

Textiles, Donghua University, No. 2999 North Renmin Road, Songjiang, Shanghai,
201620, China. E-mail: xhqin@dhu.edu.cn

Department of Biomedical Engineering, Cornell University, Ithaca, NY, 14853, USA.

E-mail: cc62@cornell.edu

c
Department of Fiber Science and Apparel Design, Cornell University, Ithaca, NY,
14853, USA

2286 | J. Mater. Chem. B, 2015, 3, 22862294

2.
2.1

Experimental
Materials

L-Lysine

monohydrochloride (Lys), p-toluenesulfonic acid

monohydrate (TosOH$H2O), adipoyl chloride, sebacoyl

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chloride, succinyl chloride, 1,4-butanediol, (Alfa Aesar, Ward

Hill, MA) and p-nitrophenol (J. T. Baker, Phillipsburg, NJ) were
used without further purication. Triethylamine (TEA) from
Fisher Scientic (Fairlawn, NJ) was dried via reuxing with
calcium hydride followed by distillation. Other solvents, such as
benzene, toluene, ethyl acetate, acetone, N,N-dimethylacetamide (DMAc) and dimethyl sulfoxide (DMSO) were purchased
from VWR Scientic (West Chester, PA) and were puried using
standard methods before use. Trypsin (Type IX-S, from bovine
pancreas, lyophilized power, 13 00020 000 BAEE units per mg
protein), hydralazine, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) were
purchased from VWR Scientic (West Chester, PA). 5-(and 6)Carboxyuorescein succinimidyl ester (NHS-uorescein, 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, the di-p-toluenesulfonic acid salt of bis-Llysine ester monomer (Lys-4) was prepared as one example to
illustrate the Lys-x monomers library. Typically, L-lysine
(0.132 mol), p-toluenesulfonic acid monohydrate (0.132 mol),
and 1,4-butanediol (0.06 mol) in 250 mL of toluene were placed
in a ask equipped with a DeanStark apparatus, a CaCl2 drying
tube and a magnetic stirrer. The solidliquid reaction mixture
was heated at 90  C and reux for 16 hours until 4.3 mL
(0.24 mol) of water was collected.
The reaction mixture was then cooled to room temperature,
ltered and dried, and nally puried by dissolving in isopropyl
alcohol at 40  C and cooling the solution to 20  C overnight for
the precipitation of the di-p-toluenesulfonic acid salt of bis-Llysine ester from isopropyl alcohol. This process was repeated
three times. Isopropyl alcohol was changed every time aer
precipitation, the white sticky mass was dried in vacuo at 40  C
for 24 hours. The nal product was a white powder and the yield
was 7080%. This new Lys-4 monomer diers from the previously published Lys-based monomers in two ways: protection
and deprotection during the synthesis are not required, and the
availability of four amine groups in each monomer instead of
one functional carboxylic acid or amine group.31,32

Journal of Materials Chemistry B

(4.5 eq. to Lys-4) was added. The above mixed solution was
placed in an 80  C oil bath for a specic time and macrogel
formation occurred, i.e., polymerization and crosslinking reactions occurred simultaneously. The resultant macrogels were
carefully removed from the vials and washed several times with
DMSO and acetone, and then with distilled water to remove any
residual chemicals. The distilled water was replaced periodically. Aer this purication 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.
2.5

Fourier transform infrared (FTIR) spectroscopy

The FTIR spectra of the monomers and macro hydrogels were

recorded on a spectrophotometer (Perkin-Elmer Magna-IR560
Spectrometer) to characterize the chemical structures of the Lys4 monomer and its hydrogels. The samples were ground into
powders, compressed into KBr discs and the FTIR spectra
recorded over the wavenumber range of 5504000 cm1. FTIR
spectra were then obtained with a PerkinElmer (Madison, WI)
Nicolet Magana 560 FTIR spectrometer with Omnic soware for
data acquisition and analysis.
2.6

Interior morphology of macrogels

Scanning electron microscopy (SEM) was employed to analyze

the interior morphological structure of the Lys-4 macrogels as a
function of the precursor feed ratio before and aer biodegradation. Typically, aer being incubated in distilled water at
room temperature for 3 days to reach its equilibrium swelling,

2.3 Synthesis of the monomers of di-p-nitrophenyl esters of

the dicarboxylic acids
The synthesis of the di-p-nitrophenyl succinate (NSu), di-pnitrophenyl adipate (NA) and di-p-nitrophenyl sebacate (NS)
monomers are described in the literature31 and are illustrated in
Fig. 1a.
2.4 Fabrication of lysine based PEA polymers and their
macrogels
The Lys-based biodegradable and pure AA-PEA polymers and
their macrogels were prepared by a direct polycondensation
reaction of Lys-4 monomer and p-nitrophenyl diester monomers at 80  C in DMAc. In brief, NS (or NA, NSu) and Lys-4
monomers at a feed molar ratio of 1.5 (or 2) were added to a vial
and dissolved in DMAc at 50  C, and then triethylamine (TEA)

This journal is The Royal Society of Chemistry 2015

(a) Chemical structure of the di-p-nitrophenyl monomers. (b)

Chemical structure of the Lys-x monomer. (c) Illustration of the
chemical structure for the hydrogel. The Lys-x part in the structure
indicates the monomer shown in (b), p-toluenesulfonic acid and the
hydrochloride salt of lysine diester. The wavey lines represent the AAPEA chains.

Fig. 1

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the macrogel was then gently removed and immediately transferred into liquid nitrogen to freeze and retain the swollen
structure. The samples were subsequently freeze-dried for three
days in a Virtis (Gardiner, NY) freeze drier in vacuo at 50  C.
For the preparation of the particles generated from the biodegraded macrogels for SEM observation, the solution medium
in which the Lys-based PEA macrogel was immersed was
removed at pre-determined periods of immersion and placed
onto a glass coverslip, dried overnight in a fume hood, and
nally xed on aluminum stubs and coated with gold for
30 seconds prior to SEM observation with a Hitachi (Mountain
View, CA) S4500 SEM instrument. Image analyses of SEM data
were performed using the public domain NIH image program.
2.7

Swelling kinetics of Lys-based PEA macrogel

Ws  Wd
 100%
Wd

(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 Lys-based macrogels was performed on a DMA Q800 Dynamic Mechanical Analyzer (TA
Instruments Inc., New Castle, DE) in controlled force mode.
The swollen macrogels samples (disc of 1.2 cm diameter) were
submerged in distilled water and mounted between a movable
compression probe (diameter 15 mm) and the uid cup. A
compression force from 0.01 to 0.05 or 0.30 N (depending on the
gel strength) at a rate of 0.02 or 0.05 N min1 was applied at
room temperature. The compression elastic modulus (E) of the
swollen macrogel was calculated from the plot the compressional stress versus strain.
2.9 In vitro enzymatic biodegradation of the Lys-based PEA
macrogel
The biodegradation of Lys-based 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 buer (pH 7.4,
0.1 M) with trypsin at a concentration of 0.1 mg mL1. A pure
PBS buer was used as the control. The vial was then incubated
at 37  C. The incubation media were refreshed daily in order to
maintain the enzymatic activity. At predetermined immersion
durations, the macrogel samples were removed from the incubation medium, and then washed three times with PBS, and
then lyophilized in vacuo with a FreeZone Benchtop and
Console Freeze Dry System (Model 7750000, LABCONCO Co.,
Kansas City, MO) at 48  C for 72 hours to a constant weight.

2288 | J. Mater. Chem. B, 2015, 3, 22862294

Wl %

W0  Wt
W0

(2)

where Wl is the percentage weight loss, W0 was the original

weight of the dried lysine based macrogel sample before
immersion, and Wt was the dried lysine based macrogel sample
weight aer an incubation time t. The weight loss data from the
average of three specimens was recorded.
To observe the nanogel generated from the macrogel
biodegradation in the enzyme solution at a predetermined time,
a few drops of the biodegradation solution were collected and
placed on silicon slides for SEM characterization.
2.10

The swelling kinetics of the Lys-based macrogels were measured

at room temperature (25  C). The dried Lys-based macrogel
were weighed and immersed in 10 mL of distilled water at room
temperature for pre-determined intervals; they were then
removed and the water on the macrogel surface gently wiped
using wet lter paper and weighed until there was no further
weight change. The swelling ratio (Q) was calculated as follows:
Q

The degree of biodegradation was estimated from the weight

loss of the macrogels based on the following equation:

Fluorescein microscopy

The nanogels generated from the biodegraded Lys-based

hydrogels were modied by coupling a uorescence dye for
visual purposes in the subsequent biological studies. In brief,
aer the macro hydrogel was biodegraded for 5 days in 0.1 mg
mL1 trypsin medium, the biodegradation solution was
collected and dialyzed through a dialysis tube (MW cuto
100 000) to remove the trypsin enzyme. Aer dialysis, predetermined amounts of NHS-uorescein were added into the
dialyzed solution containing the nanogel and the solution pH
adjusted to 5.0. NHS-uorescein was dissolved in DMSO, and
then added to the above nanogel solution, the reaction lasted
for 12 hours with stirring. The solution was collected and dialyzed against di-water using a dialysis tube (MW cuto 12 000)
to remove the unreacted NHS-uorescein and solvent DMSO.
Finally, the resulting solution was free dried using a freeze drier
to collect the dye-tagged Lys-based PEA nanogel. To obtain the
image of the NHS-uorescein attached nanogel, the freeze-dried
nanogel sample was dispersed in a PBS solution, sonicated for
10 min, and then observed by uorescence microscopy. Fluorescence microscopy was performed using a confocal uorescence microscope (C1-si, Nikon, Japan) tted with standard
FITC and Texas Red lter sets.
2.11 Nanogel size and zeta potential measurement and size
distribution
The size, size distribution and zeta potential of the Lys-based
PEA nanogel were determined using a Zeta sizer (Nano ZS,
Malvern Instruments). The nanogel contained aqueous solution
(200 mg L1) was passed through a 0.45 mm pore size lter prior
to measurement.
2.12 Drug loading in Lys-based PEA nanogel and in vitro
release
To investigate the DOX loading in the Lys-based PEA nanogel,
Lys-4, NA monomers and DOX were dissolved in DMAc and the
DOX-impregnated Lys-based PEA hydrogels were fabricated
using the procedure described towards the fabrication of lysine
based PEA polymers and their macrogels. In a typical case,
10 mg of DOX, 1.0 g of Lys-4 and 0.85 g of NA were dissolved in
DMAc and heated to 80  C for 3 min to form a DOX-impregnated

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Journal of Materials Chemistry B

Lys-based PEA macrogel. The resultant macrogel was immersed

in acetone, DMSO, and then di-water to remove any unreacted
regents. The macrogel biodegradation procedure was the same
as that described previously in the in vitro enzymatic biodegradation of lysine based macrogel with biodegradation duration
of 6 days in 0.1 mg mL1 trypsin solution. The obtained Lysbased PEA nanogel containing DOX were dialyzed in a dialysis
tube (MW cuto at 100 000 g mol1) for one day. Aer dialysis,
the dialysis tube was directly immersed into 400 mL PBS solution (pH 7.4, 0.1 M). Aliquots of 3 mL were subsequently
withdrawn from the PBS periodically at 37  C for predetermined
time. Aer each sampling, 3 mL of fresh PBS solution was added
back to the dialysis tube to maintain a constant volume of
solution. The amounts of DOX released from the nanogel were
measured using UV absorbance at 497 nm of the 3 mL aliquot
removed at predetermined periods (the calibration curve of
DOX is not shown).
To determine the drug entrapment eciency and drug
loading eciency of the Lys-based nanogel, the amounts of
DOX released from the preloaded Lys-4 based macrogel during
the swelling was tested (M1). Additionally, the amounts of DOX
released in the course of biodegradation and dialysis of the
nanogel were calculated (M2). The amount of DOX was
measured using the UV-vis absorbance at 497 nm according to
the calibration curve. The nal amounts of DOX loaded into the
nanogel were calculated using:
ML M0  M1  M2

optical density (OD) was measured at 570 nm with a Microplate

Reader Model 550 (BIO-RAD, USA). The cell viability was
calculated using the following equation. Cell viability
ODtreated/ODcontrol, where ODcontrol was obtained in the absence
of nanogels (DMEM medium only) and ODtreated was obtained
in the presence of nanoparticles with or without DOX. All cell
viability data was normalized against the blank controls (no
nanogels and no DOX).
2.14 Cell internalization of 4-Lys-4-FITC biodegradation
nanogel
Hela cells were used for the cell internalization study of FITCtagged Lys-based PEA [4-Lys-4 (1.5 : 1)] nanogel with or without
DOX [4-Lys-4 (1.5 : 1)-FITC]. Hela cells were cultured on LabTek
chamber slide dishes (2.0  104 cells per plate) to grow to about
70% conuence. The culture medium was then removed and
1 mL of fresh DMEM containing 4-Lys-4 (1.5 : 1)-FITC nanogel
(1 mg mL1) with or without DOX was added to each well. All the
cells were incubated in the plates containing DMEM and polymers for 6 h, 12 h and 24 h at 37  C, respectively. Then, the
medium was removed and the cells were washed three times
with PBS solution. Then, 1 mL of PBS solution was added into
each well. The uorescence images of the cells were observed
under excitation at 488 nm using confocal laser scanning
microscopy (CLSM) (Leica TCS SP2AOBS, Germany).

(3)

3.

where M0 is the initial amounts of DOX preloaded into the

macrogel during macrogel formation.
The drug entrapment eciency (DEE) and drug loading
eciency (DLE) in the biodegraded nanogel were dened as
follows.

3.1

2.13

DEE

Mass of loaded drug in nanogel

 100%
Mass of drug fed initially

(4)

DLE

Mass of loaded drug in nanogel

 100%
Mass of drug loaded nanogel

(5)

In vitro cytotoxicity of the Lys-based PEA nanogels

200 mL of Hela cells in DMEM medium with a cell concentration

of 1.00  104 cells per mL were added to each well in a 96-well
plate. The number of Hela cells in each well is 2.0  103. Aer
incubation for 24 h in an incubator (37  C, 5% CO2), the culture
medium was replaced with 200 mL of DMEM containing the
FITC-labeled 4-Lys-4 nanogel [4-Lys-4 (1.5 : 1)-FITC] or DOXloaded 4-Lys-4 (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
4-Lys-4 (1.5 : 1)-FITC nanogel or DOX-loaded 4-Lys-4 (1.5 : 1)FITC nanogel were replaced with fresh DMEM and 20 mL of MTT
solution (5 mg mL1) was added to the Hela cells in each well in
the 96-well plate. Aer incubation for 4 h, 200 mL of DMSO was
added and the samples shaken at room temperature. The

This journal is The Royal Society of Chemistry 2015

Results and discussion

Synthesis of the Lys-x monomers and hydrogels

Polymerization occurred between lysine monomers and diacid

based monomers (NA, NS) (Fig. 1a and b) via a single step
polycondensation reaction (Fig. 1c). The 1H NMR spectrum of
the lysine monomer is illustrated in Fig. 2a. The chemical shi
d at 8.34 ppm was attributed to amine group attached onNH2TosOH (d), d at 8.02 ppm (hydrogen chloride attached to
amine group, (l)) d at 7.75 and 7.14 ppm (the phenyl group
of TosOH attached to amine group, (b and c)), d at 4.15 ppm ((e)
C]OOCH2), d at 3.81 ppm ((g) C]OOCHNH2), d at 2.75
ppm ((k) CH2NH2), d at 2.21 ppm ((a) phenyl-CH3), d at 1.78
ppm ((j) CH2CH2CH2), d at 1.68 ppm ((h) CHCH2CH2),
d at 1.59 ppm ((f) CH2CH2CH2) and d at 1.51 ppm ((i) CH2
CH2CH2). FTIR: 3640 cm1 (NH2, wide) and 1745 cm1
(C]OO). The nal products aer polymerization could be
polymers or crosslinked polymers (macro gels), mainly
depending on the monomer ratio (Fig. 1c). The formation of the
hydrogels was demonstrated by the FTIR spectra shown in
Fig. 2c, the characteristic bands at 1742 cm1 attributed to the
C]O stretch; 1640 cm1 attributed to amide I; and 1560 cm1
attributed to amide II. These corresponding characteristic
bands at 1742 cm1, 1640 cm1 and 1560 cm1 were observed in
the 4-Lys-4 (1.5 : 1), 4-Lys-4 (2 : 1), 8-Lys-4 (1.5 : 1) and 8-Lys-4
(2 : 1) hydrogels. It can be veried that hydrogels were formed
via the condensation reaction of the amino groups in Lys-4 and
the carbonyl groups of NA or NS. As demonstrated in Fig. 2b, the
characteristic bands of 3300 cm1 were attributed to NH
stretch with the corresponding strength of 4-Lys-4 (1.5 : 1) being

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larger than that of 4-Lys-4 (2 : 1) due to the dierent feed ratio of

NA to Lys-4. As for the former, the feed ratio of NA to Lys-4 is
1.5 to 1, that indicates there are more free amino groups in the
hydrogels, the same case was observed in the 8-Lys-4 (1.5 : 1)
and 8-Lys-4 (2 : 1) hydrogels. Below the gelling point ratio,
soluble polymers will be formed and above that point, the gel
will form. Aer optimization, it was found that the gel point is
mainly aected by the reaction temperature, monomer ratio,
and concentration of the monomer (Table 1). When compared
to the reported PEA 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 for tedious protection and deprotection procedures of the
amino acids that is costly, time consuming and results in low
yields,31 (3) double bond free gelation and (4) a hydrogel having
100% pure PEA component. Subsequently, a 100% pure lysine
based PEA hydrogel library was developed with the detailed gel
synthesis protocol reported in the study. Here is one simple
example for 4-Lys-4 (x-Lys-y, where x and y represent the
methylene in the diacid monomer and diol monomer, respectively) gel: 4-Lys-4 gel was prepared by mixing NA and Lys-4 with
a molar ratio of 2 : 1 for 2.0 min at 80  C.
The polymerization/gelation rate of Lys-4 with NS is faster
than with NA in DMAc at 80  C, i.e., 2 min vs. 4 min. The
polymerization/gelation of Lys-4 monomers with Nsu monomer
(x 1 in Fig. 2b) under the same reaction conditions as with NA

Paper

or NS, however, was unsuccessful. This was due to the presence

of less methylene or activity in the Nsu monomer that form a
short spacer when polycondensation occurred, which made it
dicult to fabricate a hydrogel.
3.2 Swelling kinetics of the Lys-based PEA macrogel and
mechanical properties
Fig. 1c is an illustration of the hydrogel crosslinking formed
upon reaction of the monomer A and monomer B used to form
the hydrogel. Interestingly, it was found that the polymerization/gelation rate and gel structure were determined by the
monomer structure under xed reaction conditions. For
example, Lys-4 shows a faster gelation rate when reacting with
NS than with NA in DMAc at 80  C, i.e., 2.0 min vs. 4.0 min. The
polymerization/gelation of Lys-4 monomers with the Nsu
monomer (x 1 in Fig. 1b) under the same reaction conditions,
however, was only able to form a very so hydrogel or viscous
mixture. This may be due to the weaker reactivity or presence of
less methylene in the Nsu monomer, which made it dicult to
fabricate a hydrogel. The modulus of the resultant hydrogels
was also aected by the monomer structure: higher x or y would
cause a higher modulus (Table 2). For example, the 8-Lys-4
hydrogel had a signicantly higher compression modulus
(more than 25 times) than the 4-Lys-4 hydrogel with the same
monomers feed ratio, i.e., 670  53 kPa vs. 25  4.5 kPa,
respectively (Table 2).
To successfully obtain nanogels via an enzyme biodegradation strategy, one of the key factors is the three dimensional
structure of the 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 Lys-x (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 well-dened, three-dimensional porous
and completely connected network structures with an average
wall thickness of around 1.04.0 mm and average pore size
ranging from 4.0 to 10.0 mm. It was found that the x, y and m to n
ratio aects the pore size and wall thickness. For example, as
shown in Fig. 2e, the average pore size of the hydrogel were

Table 1

Fig. 2 (a) 1H NMR spectrum of the Lys-4 monomer (300 MHz, DMSOd6). (b) Swelling kinetics of the Lys-4 based macrogels in distilled water
at 25  C. (c) FTIR spectra of the lysine based hydrogel. (A) 4-Lys-4
(2 : 1); (B) 4-Lys-4 (1.5 : 1); (C) 8-Lys-4 (2 : 1); (D) 8-Lys-4 (1.5 : 1). 1742
cm1 attributed to the C]O stretch; 1640 cm1 attributed to amide I;
and 1560 cm1 attributed to amide II. (d) Optical images of the
hydrogels. (A) 8-Lys-4 (1.5 : 1); and (B) 4-Lys-4 (1.5 : 1). The molar ratio
of 1.5 : 1 indicates NA or NS to the Lys-4 monomer. (e) SEM images of
the Lys-4 based hydrogels before degradation. (A) 8-Lys-4 (2 : 1); (B)
8-Lys-4 (1.5 : 1); (C) 4-Lys-4 (2 : 1); and (D) 4-Lys-4 (1.5 : 1).

2290 | J. Mater. Chem. B, 2015, 3, 22862294

Preparation of Lys-4 based macrogels

Sample code

NA

NS

Lys-4

Lys-6

Lys-8

DMAc

Et3N

4-Lys-4 (1.5 : 1)
4-Lys-4 (2 : 1)
8-Lys-4 (1.5 : 1)
8-Lys-4 (2 : 1)
4-Lys-6 (1.5 : 1)
4-Lys-6 (2 : 1)
4-Lys-8 (1.5 : 1)
4-Lys-8 (2 : 1)
8-Lys-6 (1.5 : 1)
8-Lys-6 (2 : 1)
8-Lys-8 (1.5 : 1)
8-Lys-8 (2 : 1)

0.76 g
1.02 g

0.67 g
0.95 g
0.65 g
0.89 g

0.87 g
1.16 g

0.81 g
1.08 g
0.76 g
1.01 g

1.0 g
1.0 g
1.0 g
1.0 g

1.0 g
1.0 g

1.0 g
1.0 g

1.0 g
1.0 g

1.0 g
1.0 g

2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0

0.65 g
0.65 g
0.65 g
0.65 g
0.65 g
0.65 g
0.65 g
0.65 g
0.65 g
0.65 g
0.65 g
0.65 g

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Journal of Materials Chemistry B

6.0 mm and 4.0 mm for 4-Lys-4 (1.5 : 1) (1.5 : 1 is the molar ratio
of monomer NA to Lys-4) and 4-Lys-4 (2 : 1), and 10.0 mm and
8.0 mm for 8-Lys-4 (1.5 : 1) and 8-Lys-4 (2 : 1), respectively. The
4-Lys-4 hydrogels had thinner cell walls than the 8-Lys-4
hydrogels, which indicates 8-Lys-x hydrogels had a more
compact structure. In addition, the average pore size decreased
with an increase in x. With an increase in amide group density
formed in the hydrogel, i.e., an increase in cross-linking density,
the average pore size decreased, which is consistent with the
swelling ratio and mechanical data of the macro hydrogels
(Fig. 2b).
3.3 In vitro enzymatic biodegradation of Lys-based PEA
macrogel
The enzyme biodegradation mechanism of the cross-linked
lysine based hydrogels was subsequently investigated using a
hydrolytic enzyme with a series of concentrations in PBS buer
solutions. The weight loss, size distribution and SEM were used
to characterize and monitor the biodegradation progress of the
hydrogels. The hydrolytic enzyme, trypsin, is utilized here as a
model enzyme because of its general degradation capability. In
addition, we have focused on the material related properties in
this study. The results indicated that the degradation behavior
is aected by a variety of factors, including crosslinking density,
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 the lysine based hydrogel depended on the crosslinking density of the amide groups and the hydrophobicity of
the hydrogels. For the 4-Lys-4 (1.5 : 1) hydrogel immersed in
PBS solution without trypsin, its weight loss was only 5.0 wt%
aer 10 days, which indicates the contribution from the
hydrolytic degradation of the buer was very small or negligible.
Therefore, the degradation contribution is mainly due to
enzyme attack. The biodegradation rate of the hydrogel could
be regulated by tuning the crosslinking density. For example,
4-Lys-4 (1.5 : 1) is faster than 4-Lys-4 (2 : 1), which was ascribed
to the lower crosslinking density of the amide groups in the
hydrogels. With an increase in the ratio of the monomer NA to
Lys-4, the hydrogel has a higher crosslinking density of the
amide groups. A similar trend was observed in the 8-Lys-4
hydrogels. In general, the degradation rate of the hydrogel is
related to several network parameters such as the number of
crosslinkers per backbone chain, molecular weight of the
backbone unit and the proportion of biodegradable groups in
the main and side chains.3335 Therefore, the biodegradation

Table 2

Compressive modulus of the Lys-4 based macrogels

Sample code

Monomer molar feed ratio

Compressive
modulus (kPa)

4-Lys-4 (1.5 : 1)
4-Lys-4 (2 : 1)
8-Lys-4 (1.5 : 1)
8-Lys-4 (2 : 1)

NA : Lys-4 1.5 : 1
NA : Lys-4 2 : 1
NS : Lys-4 1.5 : 1
NS : Lys-4 2 : 1

25  4.5
46  6.1
670  53
1350  126

This journal is The Royal Society of Chemistry 2015

rate decreased with an increase in crosslinking density. It was

found that the hydrogel with a higher swelling ratio had a faster
biodegradation rate in our study, which is consistent with the
above rules. Aer screening, 8-Lys-4 and 4-Lys-4 gels were
chosen for the following study.
SEM was used to monitor the morphology change of the
swollen 8-Lys-4 and 4-Lys-4 hydrogels during enzymatic
biodegradation. On the second day of biodegradation, there are
a lot of regular microspheres on the hydrogel surface and in the
hydrogel networks, and the average size ranges from 0.5 mm to
4.0 mm (Fig. 3a). This is the rst time that the synthesized
hydrogel was reported to be able to produce microspheres
(microgels) aer being treated with an enzyme. With the prolonged biodegradation time, the enzymatic biodegradation of
the hydrogel network continues and the average size of the
microsphere was decreased to below 3.0 mm aer 2 days. The
morphology of the microspheres became less regular and some
of the microspheres linked like a string of beads, indicating the
1st stage biodegradation was incomplete (Fig. 3b). Moreover,
the average pore size of the hydrogels increased during the
biodegradation process. As shown in Fig. 3c, the average pore
size of the 8-Lys-4 (1.5 : 1) and 8-Lys-4 (2 : 1) was around
10.0 mm and 6.0 mm, respectively, and the size for 4-Lys-4

Fig. 3 (a) SEM images of the lysine based macrogels with an enzyme
trypsin concentration of 0.1 mg mL1 in PBS buer (pH 7.4, 0.1 M) after
1 day of biodegradation. (A) 4-Lys-4 (1.5 : 1); (B) 4-Lys-4 (2 : 1); (C) 8Lys-4 (1.5 : 1); and (D) 8-Lys-4 (2 : 1). (b) SEM images of the lysine
based hydrogel with an enzyme trypsin concentration of 0.1 mg mL1
in PBS buer (pH 7.4, 0.1 M) after 3 days of biodegradation. (A) 4-Lys-4
(1.5 : 1); (B) 4-Lys-4 (2 : 1); (C) 8-Lys-4 (1.5 : 1); and (D) 8-Lys-4 (2 : 1).
(c) SEM images of the lysine based macrogels with an enzyme trypsin
concentration of 0.1 mg mL1 in PBS buer (pH 7.4, 0.1 M) after 4 days
biodegradation. (A) 4-Lys-4 (1.5 : 1); (B) 4-Lys-4 (2 : 1); (C) 8-Lys-4
(1.5 : 1); and (D) 8-Lys-4 (2 : 1). Microgels and nanogels are invisible
because most of them have been washed out with PBS. (d) SEM images
of the lysine based macrogels with an enzyme trypsin concentration of
0.1 mg mL1 in PBS buer (pH 7.4, 0.1 M) after 5 days of biodegradation. (A) 4-Lys-4 (1.5 : 1); (B) 4-Lys-4 (2 : 1); (C) 8-Lys-4 (1.5 : 1); and
(D) 8-Lys-4 (2 : 1). Microgels and nanogels are invisible because most
of them have been washed out with PBS.

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(1.5 : 1) and 4-Lys-4 (2 : 1) was around 12.0 mm and 10.0 mm,

respectively. Aer 4 days, the average pore size of the 8-Lys-4
(1.5 : 1), 8-Lys-4 (2 : 1) was around 20.0 mm and 15.0 mm,
respectively; the average size for 4-Lys-4 (1.5 : 1) and 4-Lys-4
(2 : 1) was 30.0 mm and 40.0 mm, respectively, and the regular 3D structure of the hydrogels was almost broken (Fig. 3d). The
zeta potential of the 4-Lys-4 (1.5 : 1) and 4-Lys-4 (2 : 1) nanogel
was 14.5 mV and 9.6 mV, respectively, indicating net free
primary amine groups on the microsphere surface.
At 5 days, the microspheres on the hydrogel surface were
partially biodegraded and some of the broken microspheres
were 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 their size
decreased to about 200 nm (Fig. 4b). Aer 8 days biodegradation, the size of the obtained nanogels was about 50 nm with a
narrow size distribution (Fig. 4c). The size of the micro and
nano gels in PBS solution were characterized by DLS. The
enzyme trypsin was removed by dialysis (cuto weight 100 000).

3.4 Drug loading in the Lys-based PEA nanogels and in vitro

release
The biodegradation behavior of the drug loaded Lys based
macro hydrogels were further examined using the DOX as a
model drug. The hydrogels were biodegraded using 0.1 mg

Paper

mL1 trypsin PBS solution, as described in the previous section.

The DOX was preloaded into the hydrogels during the macro
hydrogel fabrication. In this study, for a 5.0 wt% drug added,
the drug entrapment eciency (DEE) and drug loading eciency (DLE) was 51.6% and 2.6%, respectively. The DOX loaded
nanogels were obtained and chosen for a release study aer
5 days of degradation. The resultant nanogels were washed with
PBS before testing the DOX release. The yield of the above 4-Lys4 (1.5 : 1) nanogel was 18.6%. As demonstrated in Fig. 5b, at a
time of 5 hours, the cumulative DOX release from the nanogels
in both PBS solution (pH 7.4, 0.1 M) and enzyme trypsin solution (0.1 mg mL1) was about 10.0%. However, the release of
DOX became a virtually constant rst order rate aer 15 hours
over a 10 day period for nanogels in PBS solution (pH 7.4, 0.1 M,
0.1 mg mL1 trypsin).
With the degradation of the ester and amide bonds of the
hydrogel, the hydrophilic lysine segments of the nanogel dissolved and diused into the buer 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
time (1st order). Aer degrading the 4-Lys-4 (1.5 : 1) nanogel,
the release behavior came to an end. This indicates that the
release of DOX was mainly controlled by the dissolution and
dissociation of the 4-Lys-4 (1.5 : 1) nanogels rather than by a
diusion mechanism.
3.5 Cell internalization of the 4-Lys-4-FITC biodegradation
nanogel
Monomer Lys-4 has 4 free amine groups, when the molar ratio
of NA to the lysine based monomer Lys-4 is 1.5 : 1, the resultant
4-Lys-4 (1.5 : 1) hydrogel has free amine groups aer hydrogel
formation compared to the 4-Lys-4 (2 : 1) hydrogel. The free
amine groups endow the nanogel a positive charge for in vitro

Fig. 4 (a) The weight loss of the lysine based hydrogels during
degradation. The hydrogels were immersed in PBS buer (pH 7.4,
0.1 M) with trypsin at a concentration of 0.1 mg mL1 and pure PBS
used as a control. (b) SEM images of the lysine based 4-Lys-4 (1.5 : 1)
macrogels with an enzyme trypsin concentration of 0.1 mg mL1 in
PBS buer (pH 7.4, 0.1 M) after (A) 6 days of biodegradation; and (B)
7 days of biodegradation. (C) DLS of the 6 day biodegradation nanogel
and (D) DLS of the 7 day biodegradation nanogel. (c) SEM images of the
lysine based 4-Lys-4 (1.5 : 1) hydrogels with an enzyme trypsin
concentration of 0.1 mg mL1 in PBS buer (pH 7.4, 0.1 M) after (A)
8 days of biodegradation and (B) 9 days of biodegradation. (C) DLS of
the 8 day biodegradation nanogels and (D) DLS of the 9 day biodegradation nanogels.

2292 | J. Mater. Chem. B, 2015, 3, 22862294

(a) CLSM images of the biodegradation particles: (A) em 595 nm

for DOX; (B) em 515 nm for FITC and (C) a physically overlaid image of
panels A and B. (b) Cumulative release of DOX from the 4-Lys-4
(1.5 : 1) hydrogel biodegraded nanogels at 37  C in PBS solution (pH
7.4, 0.1 M) at a trypsin concentration of 0.1 mg mL1. (c) Cytotoxicity of
the 4-Lys-4 (1.5 : 1)-FITC nanogels, DOX loaded 4-Lys-4 (1.5 : 1)-FITC
nanogels and free DOX at dierent concentrations. Control for free
DOX indicates no DOX for cell culture.
Fig. 5

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multifunctional L-lysine based diester monomer was specially

developed and a corresponding crosslinking method used to
chemically fabricate a new family of pure amino acid-based
poly(ester amide) hydrogels with desired chemical structures.
For some screened macrogels, it was found that spherical
nanogels with controllable size were formed and the nal size
could be below 50 nm aer being biodegraded for 8 days. With
the abundance of free amine groups, the degraded microgel and
nanogels could easily conjugate with biologically active reagents
or dyes and demonstrate a controllable and sustained release,
therefore, expanding the biomedical applications of this gel
platform.

Acknowledgements

Fig. 6 Confocal laser scanning microscopy images of Hela cells after

6 h (A1, B1, C1, D1); 12 h (A2, B2, C2, D2); 24 h (A3, B3, C3, D3); and free
DOX (A0, C0, D0) incubated with DOX loaded 4-Lys-4 (1.5 : 1)-FITC
nanogels and free DOX (20 mg mL1) as the control in the DMEM
medium at 37  C. (A1, A2, A3, A0): bright eld images, B1, B2, B3, B4:
confocal green uorescence images; C1, C2, C3, C4: confocal red
uorescence images; D1, D2, D3, D4: merge.

cellular uptake and chemical conjugation, such as uorescence

dye attachment. CLSM was used to investigate the 4-Lys-4
(1.5 : 1)-FITC biodegradation nanogels and DOX loaded 4-Lys-4
(1.5 : 1)-FITC nanogel's uptake by HeLa cells and their subsequent accumulation. The uoresence dye, FITC, was chemically
conjugated to the nanogel upon reaction with its amine groups
and was used to track the nanogel in the cell for imaging. Aer
6 h of incubation with the DOX loaded 4-Lys-4 (1.5 : 1)-FITC
nanogels suspension (1 mg mL1) at 37  C, uorescence was
observed in HeLa cells (Fig. 6). With the incubation time
increasing, a higher uorescence intensity was observed, as
shown in Fig. 6B2. When the HeLa cells grew to 24 h with the
DOX loaded 4-Lys-4 (1.5 : 1)-FITC nanogels, a much higher
intensity of the red uorescence was seen inside the Hela cells.
This is consistent with the MTT results of the DOX loaded 4-Lys4 (1.5 : 1)-FITC nanogels, as shown in Fig. 5c. The cytotoxicity
increased with the increased concentration of DOX loaded
4-Lys-4 (1.5 : 1)-FITC nanogels. When the concentration of DOX
loaded 4-Lys-4 (1.5 : 1)-FITC nanogels is 0.25 mg mL1, the cell
viability is less than 20%. When the concentration is 0.5 mg
mL1, the cell viability decreased to 10%. Finally, at a concentration of 1.0 mg mL1, the cell viability decreased to 8%.

4. Conclusions
In conclusion, a novel strategy was utilized to develop functional microgels and nanogels from macrogels using the
controlled enzyme biodegradation of the screened macrogels.
To achieve the goal of nanogel fabrication, a new

This journal is The Royal Society of Chemistry 2015

This research is supported by a grant from the Rebecca Q.

Morgan Foundation. The research is also supported by the
Fundamental Research Funds for the Central Universities and
sponsored by the Shanghai Pujiang Program 14PJ1400300. The
authors are grateful for the Nanobiotechnology Center (NBTC)
for using the research facilities in Cornell University for cell
related work.

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