Diffusion-Controllable Biomineralization Conducted in Situ in Hydrogels Based on Reversibly Cross-Linked Hyperbranched Polyglycidol

Article abstract of DOI:10.1021/acs.biomac.7b01071

We present biocompatible hydrogel systems suitable for biomineralization processes based on hyperbranched polyglycidol cross-linked with acrylamide copolymer bearing carbonyl-coordinated boronic acid. At neutral pH, diol functional groups of HbPGL react w

Full text of DOI:10.1021/acs.biomac.7b01071

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Article
Diffusion-controllable biomineralization conducted in situ in hydrogels
based on reversibly cross-linked hyperbranched polyglycidol
Mateusz Gosecki, Slawomir Kazmierski, and Monika Gosecka
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01071 • Publication Date (Web): 05 Sep 2017
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Diffusion-controllable biomineralization conducted in situ in hydrogels based on reversibly
cross-linked hyperbranched polyglycidol
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Mateusz Gosecki†, Slawomir Kazmierski †, Monika Gosecka†*
†
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences,
ul. Sienkiewicza 112, 90-363 Lodz
*
mdybko@cbmm.lodz.pl; phone number: +48 42 6803235
Abstract
We present biocompatible hydrogel systems suitable for biomineralization processes based
on hyperbranched polyglycidol cross-linked with acrylamide copolymer bearing carbonyl-
coordinated boronic acid. At neutral pH, diol functional groups of HbPGL react with boronic
acid of polyacrylamide to generate 3D network in water by the formation of boronic ester
cross-links. The dynamic associative/dissociative characteristics of the cross-links makes the
network reversible. The presented hydrogels display self-healing properties and are
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injectable, facilitating gap filing of bone tissue. The H HR MAS DOSY NMR studies reveal that
acrylamide copolymer plays the role of the network framework, whereas HbPGL
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macromolecules, due to their compact structure, move between reactive sites of the
copolymer. The influence of the copolymer macromolecules entanglements and overall
polymer concentrations on macromolecules mobility and stress relaxation processes is
investigated. The process of hydrogel biomineralization results from hydrolysis of 1-naphthyl
phosphate calcium salt catalysed by encapsulation in hydrogel alkaline phosphatase. The
environment of the hydrogel is entirely neutral towards the enzyme. However, the activity of
alkaline phosphatase encapsulated within the hydrogel structure is diffusion-limited. In this
article, based on the detailed characteristics of three model hydrogel systems, we
demonstrate the influence of the hydrogel permeability on the encapsulated enzyme activity
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and calcium phosphate formation rate. The H HR MAS DOSY NMR is used to monitor
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diffusion low-molecular weight compound within hydrogels, whereas P HR MAS NMR
facilitates monitoring of the progress of biomineralization in situ within hydrogels. The
results show a direct correlation between low molecular diffusivity in hydrogels and network
dynamics. We demonstrate that the morphology of in situ-generated calcium phosphate
within three model HbPGL/poly(AM-ran-APBA) hydrogels of different low molecular
permeability varies substantially from sparsely deployed large, well-defined crystals to an
even distribution within the polymers polycrystalline continuous network.
KEYWORDS
injectable, hydrogel, biomineralization, diffusion, DOSY NMR, hyperbranched polyglycidol,
polyacrylamide
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INTRODUCTION
Encapsulation of enzymes in polymer matrices is generally undertaken to protect
biomacromolecules from unfolding or aggregation, which leads to a loss of protein biological
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activity after extraction from their native environment. In addition to the storage aspect, it is
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also undertaken due to many specific applications, amongst which the catalysis of diverse
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chemical reactions is the most popular.
This approach is often more practical in
comparison to the usage of enzyme in the free state, as isolation of the desired product after
the reaction is more convenient. It also facilitates enzyme recovery for applications during
next reaction cycles. Apart from catalysis, the field of biomedical application of enzymes is of
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great importance and interest. It includes the usage of polymer hydrogel matrices enriched
with enzymes for the biomineralization process. For this goal, alkaline phosphatase may be
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entrapped within the hydrogel structure,
in which the enzyme triggers bone
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biomineralization processes by catalysing the hydrolysis of organic phosphates .
Certain features are required from hydrogel systems to be applied for enzymatically
triggered biomineralization. The polymer matrix should be bioinert, i.e., not interact with
biomolecules by covalent bonds or nonspecific interactions, which may result in protein
denaturation. Moreover, the process of biomolecules encapsulation should be carried out
under mild enough conditions (pH, temperature, among others) to preserve the biological
activity of the enzyme. Among the polymers that were applied for hydrogel formation in the
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biomineralization process were biopolymers such as chitosan, alginate, silk fibroin, and
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agarose, as well as synthetic polymers, i.e., poly(ethylene glycol), poly(vinyl alcohol),
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poly(vinyl pyrrolidone), and poly(acrylic acid),
among others. The most popular
method for enzyme encapsulation in hydrogels is its incorporation during the gelation process.
This approach ensures the homogeneous distribution of enzyme molecules within the
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hydrogel structure. However, the crossꢀlinking reaction can have detrimental effects on the
protein structure, including changes in the active centre. For example, in the case of alginate
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gelation with Ca ions, the structure of the proteins can be changed due to competing
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interactions of the carboxyl groups.
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Especially attractive hydrogels are those that are injectable and that have a structure
based on reversible dynamic chemistry ensuring healing properties. Reversible characteristics
of bonds ensures continuous reorganization of the gel structure, i.e., healing properties of the
hydrogel material due to the constant associationꢀdissociation of crossꢀlinks. These properties
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can be triggered by a specific stimulus such as temperature and pH, among others. Healing
properties are ensured by fast exchange reactions and sufficient mobility of network building
macromolecules. These properties facilitate the introduction of the hydrogel into gaps in bone
tissue. Moreover, healing properties guarantee the formation of hydrogel in one piece in the
case of any disruption during the introduction or addition of the next portion of hydrogel.
Hydrogel flow characteristics can be controlled by the strength and degree of crossꢀlinks.
Here, we present a new type of hydrogel that is suitable for biomineralization
applications, which is composed of hyperbranched polyglycidol (HbPGL) reversibly crossꢀ
linked with a copolymer of acrylamide and 2ꢀacrylamidophenylboronic acid. Interest in the
use of HbPGL in hydrogel formation results from its advantageous characteristics, such as
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biocompatibility, superhydrophilicity and antibiofouling/ protein repellent behaviours
.
The topology of HbPGL and its flexibility facilitate the compartmentalization of various
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molecules within the macromolecule structure, i.e., drugs and labels, among others.
Moreover, the presence of 1,2ꢀdiol functional groups in the terminal units of HbPGL provides
the possibility of the formation of crossꢀlinks with boronic acid in aqueous solutions and the
creation of reversible, covalent hydrogel systems. Generally, the formation of diolꢀboronic
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acid crossꢀlinks is preferred at an alkaline pH. However, Deng et al reported the possibility
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of diolꢀboronic acid crossꢀlinks formation at acidic and neutral pH values when the boron
atom is intramolecularly coordinated with an oxygen. Polyacrylamide is also a wellꢀknown
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hydrophilic and bioinert polymer.
In addition copolymers acrylamide and 2ꢀ
[31]
acrylamidophenylboronic (poly(AMꢀranꢀAPBA)) acid can be easy synthesized.
The
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presence of a carbonyl oxygen atom close to the boronic acid moiety in 2ꢀ
acrylamidophenylboronic acid ensures the intramolecular coordination with the boron atom.
In reversibly crossꢀlinked polymer networks, dynamic events, i.e., the crossꢀlink
lifetime and the mobility of macromolecules, influence the diffusion rate of compounds
embedded in the gel. Their diffusion can be modified by changes in the degree of crossꢀ
linking of the network. For example, in the case of networks based on diolꢀboronic acid crossꢀ
links, it can be achieved by changing the temperature because crosslinking is an exothermic
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process . The diffusion of reactants within the hydrogel matrices is of great importance
because it influences other processes in the network.
In this article, we investigate alkaline phosphatase ꢀ triggered calcium phosphate
formation within HbPGL/poly(AMꢀranꢀAPBA) hydrogels with respect to reagents diffusion.
We relate this dependence to the dynamics of hydrogels determined by rheological
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measurements and diffusion data. By using H HR MAS DOSY NMR, we were able to
monitor the diffusion coefficients of the hydrogel building polymer, including the influence of
temperature, as well as the diffusion coefficient of lowꢀmolecular weight probe – pꢀ
nitrophenol. This molecule, which is the product of the hydrolysis of pꢀnitrophenyl phosphate,
was also used to estimate the effective bioactivity of alkaline phosphatase within hydrogels.
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In addition, based on P HR MAS NMR spectra, we were able to monitor the process of
calcium phosphate formation in situ within the hydrogel as a result of 1ꢀnaphthyl phosphate
calcium salt hydrolysis.
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EXPERIMENTAL SECTION
Materials
The pꢀnitrophenyl phosphate disodium salt hexahydrate and 1ꢀnaphthyl phosphate calcium
salt trihydrate were used as received from SigmaꢀAldrich. Acrylamide (SigmaꢀAldrich) was
recrystallized from acetone. Glycidol, 96%, was obtained from SigmaꢀAldrich and distilled
under a vacuum (61°C; 15 mmHg). Comonomer 2ꢀacrylamidophenylboronic acid pinacol
[31]
ester was prepared according to the procedure described in Ref.
The 2,2ꢀ
azobisisobutyronitrile, AIBN (Fluka) was recrystallized from ethanol. Alkaline phosphatase
from bovine intestinal mucosa was purchased from SigmaꢀAldrich. PBS buffer (pH=7.4) was
prepared with deionized water. For NMR analysis of polymer solutions, deuterated PBS
(pD=7.4) was applied, whereas for HR MAS NMR analysis hydrogel samples were prepared
2 2
from PBS composed of H O and D O (9:1, v/v).
Synthesis of HbPGL, hyperbranched polyglycidol
Hyperbranched polyglycidol was synthesized by anionic polymerization according to the
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procedure described by Sunder . Briefly, 10% of the hydroxyl groups in 1,1,1ꢀ
tris(hydroxymethyl)propane (0.0043 mol; 0.5770 g) were converted into alcoholate form in a
reaction with sodium hydride (0.0129 mol; 0.031 g). Then, glycidol (0.4 mol; 29.60 g) was
added at a rate of 2 ml/h to a reactor equipped with a mechanical stirrer. The reactor was kept
in an oilꢀbath at 95°C for 14 h in a dry argon atmosphere. The obtained product was dissolved
in methanol. Sodium ions at alcoholate sites were exchanged for protons by passing the
polymer through a cationꢀexchange resin (Dowex 50 WX 4). The product was precipitated
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three times from methanol in acetone to remove cyclic byꢀproducts, dried under a vacuum and
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characterized by H NMR, C NMR and GPC methods (chromatograms and NMR spectra
are shown in SI in Figures S1, S2 and S3, respectively).
ꢁ ꢁ
All experiments were carried out using HbPGL with ꢀ ꢃꢄꢅꢆꢇ = 6400;ꢈꢀ /ꢀ = 1.56 and
ꢂ
ꢁ
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ꢉ ꢂ
DB (degree of branching) = 0.61. The fraction of vicinal diol in the terminal units was
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estimated on the basis of the C NMR spectrum and was approximately 34.0 mol%.
Syntheses of poly(AM-ran-APBA), poly(acrylamide-ran-2-acrylamide phenylboronic
acid) with 6.0 mol% of APBA
We have synthesized two copolymers, each one with 6.0 mol% APBA content differing in the
molecular weight by the application of different AIBN amounts. The copolymers were
prepared by conventional radical polymerization initiated with AIBN by applying acrylamide
(2 g; 28.10 mmol) and 2ꢀacrylamidophenylboronic acid pinacol ester (0.613 g; 2.24 mmol).
Polymerizations were carried out in 15 ml of DMF/dioxane mixture (5:1 v/v) at 70°C. The
syntheses were conducted for 16 hours. Each polymerization mixture was diluted in water,
and the copolymer was precipitated into acetone and dried. Next, the copolymer was
dissolved in alkaline solution of NaOH (1 wt%) and dialyzed using a 1000 MW cut off
dialysis membrane, at first against the alkaline aqueous solution and then against water, which
was changed several times to reach the neutral pH. Dialysis was necessary to hydrolyse
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pinacol boronic esters and remove released pinacol. The exemplary H NMR spectra
confirming the molar composition of copolymer are presented in SI in Figure S4 and Figure
S5. Different molecular weights of poly(AMꢀranꢀAPBA) were obtained by changing the
amount of introduced AIBN against the comonomers. For the preparation of a shorter
copolymer, COP1 (i.e., MnCOP1= 23000, M
w n
/M =1.18), the initial molar ratio of comonomers
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to AIBN was222:1 , whereas for the synthesis of the copolymer of MnCOP2= 41000, COP2
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M /M =2.16), it was 1000:1.
w n
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Preparation of HbPGL-co-/poly(AM-ran-APBA) hydrogel systems
HbPGL/poly(AMꢀranꢀAPBA) hydrogels were prepared in an independent PBS (pH=7.40)
stock solution. Solutions were mixed together to induce immediate gelation. The detailed
composition of prepared hydrogels are shown in Table 1.
Table 1. Composition of HbPGL/poly(AM-ran-APBA) hydrogels.
Hydrogel
Weight fraction HbPGL
Molar ratio of Number of -B(OH)2
of
wt%
polymers, macromolecules
diol:boronic
groups per 1 HbPGL
macromolecule
per macromolecules acid
of poly(AM-ran-APBA)
H1
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15.2: 1 COP2
14.0:1
6.5:1
6.5:1
2.0
4.4
4.4
H3A
H3B
7.0: 1 COP2
4.0:1 COP1
Preparation of hydrogels for effective activity measurements of an alkaline phosphatase
We have prepared a series of hydrogels differing in molar concentration of pꢀnitrophenyl
phosphate (substrate) to determine the maximum reaction rate of hydrolysis of organic
phosphate entrapped in the hydrogel. The concentration of substrate in the hydrogel was in the
range from 1 mmol/l to 46 mmol/l. Polymer hydrogels were prepared by dissolving each
polymer in an independent stock solution. The weight fraction of polymer components was
the same as shown in Table 1. Polyglycerol was dissolved in PBS buffer (pH=7.4) containing
pꢀnitrophenyl phosphate, whereas the solution of poly(AMꢀranꢀAPBA) in PBS buffer
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contained an alkaline phosphatase. Mixing the two solutions caused immediate gelation. The
final concentration of phosphatase in the hydrogel was 1.76 mg/ml.
Preparation of hydrogels for NMR diffusion measurements
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Polymer hydrogels were prepared by dissolving each polymer in an independent stock
solution. Polyglycerol was dissolved in the solution of pꢀnitrophenyl phosphate prepared in
PBS buffer (pH=7.4), whereas the solution of poly(AMꢀranꢀAPBA) in PBS buffer contained
an alkaline phosphatase. Then, these solutions were mixed carefully resulting in immediate
gelation. Up to 15 minutes, within all investigated hydrogels, the entire amount of introduced
pꢀnitrophenyl phosphate was hydrolysed to pꢀnitrophenol, which was used as a probe for
small molecules diffusion using the model HbPGL/poly(AMꢀranꢀAPBA) hydrogels. The final
concentration of pꢀnitrophenyl phosphate within all investigated hydrogels was 34.5 mmol/l,
whereas the final concentration of alkaline phosphatase was 1.76 mg/ml. The weight fraction
of polymer components was the same as shown in Table 1.
Preparation of hydrogels for biomineralization
The solution of naphthyl phosphate calcium salt (c=20.98 mg/ml) was prepared in PBS buffer
and filtered through a 0.2ꢀꢁm syringe membrane. Polymer hydrogels were prepared by
dissolving each polymer in an independent stock solution. Polyglycerol (0.117 g in case of H1
hydrogel; 0.055 g in case of H3A and H3B hydrogels) was dissolved in the solution of
naphthyl phosphate calcium salt (0.246 ml in case of H1 and 0.200 ml in case of H3A and
H3B hydrogels) prepared in PBS buffer (pH=7.4), whereas the solution of poly(AMꢀranꢀ
APBA), 0.0495 g in 0.154 ml PBS buffer in case of H1 hydrogel (0.150 ml PBS in case of
H3A and H3B hydrogels) contained an alkaline phosphatase. Then, these solutions were
mixed carefully resulting in immediate gelation. The rate of phosphate ester hydrolysis within
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hydrogels was monitored by P HR MAS NMR spectroscopy. The detailed composition of
the hydrogels is shown in Table 2.
Table 2. Composition of hydrogels used for in situ triggered biomineralization.
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Hydrogel
Weight
fraction
of Concentration of naphthyl
polymers, wt%
phosphate calcium salt,
mg/ml
H1_biom*
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9.11
H3A_biom*
H3B_biom*
9.11
9.11
*
The concentration of alkaline phosphatase was 1.76 mg/ml.
Rheology
Gel formation was confirmed with oscillation frequency sweep tests carried out in the linear
viscoelastic regime using a parallel plateꢀplate geometry of 8 mm diameter with a 0.3 mm gap
on a Thermoscientific HAAKE MARS 40 rheometer.
An exemplary measurement of selfꢀhealing properties based on H3A_1.76 mg/ml was carried
out in the 1 Hz oscillation time sweep test at 293 K by monitoring storage and loss moduli.
First, the sample was placed under 1% of strain, and then it was being destroyed for 5 seconds
with 300% strain, after which 1% was again applied for sample restoration. The cycle was
repeated once.
Alkaline phosphatase activity assay
As alkaline phosphatase hydrolyses organic phosphates, the usage of pꢀnitrophenyl phosphate
as a substrate facilitated monitoring the efficient biological activity of the enzyme, by the
release of pꢀnitrophenol (Scheme 1), which is a yellowꢀcoloured product with the
characteristic maximum absorbance at 405 nm. We have prepared series of each hydrogel
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type (H1, H3A and H3B) with constant concentration of alkaline phosphatase, i.e., 1.76
mg/ml and different amounts of the entrapped pꢀnitrophenyl phosphate in the range from 1
mmol/l to 46 mmol/l. The rate of product formation was determined based on the absorbance
value corresponding to pꢀnitrophenol released at 37°C after 5 minutes of incubation. After this
time, each hydrogel system was immediately dissolved in 9 ml 0.1 M NaOH solution by
vigorous shaking. Then, 0.20 ml of the analysed solution was introduced into 2.80 ml of PBS
buffer. Absorbance measurements of pꢀnitrophenol were carried using a Specord S 600
analytik jena instrument. Based on the change in absorbance, the MichaelisꢀMenten constant
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of the enzyme entrapped within the hydrogel, K and the maximum rate of the reaction, Vmax.
m
m
K , Vmax were determined with the OriginPro nonꢀlinear fit tool using the Levenberg–
Marquardt optimization algorithm. As a reference, the activity of free alkaline phosphatase in
the native form was measured by applying identical concentrations of enzyme and pꢀ
nitrophenyl phosphate.
Scheme 1. Hydrolysis of pꢀnitrophenyl phosphate into pꢀnitrophenol catalysed by alkaline
phosphatase.
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NMR analysis
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H NMR and H DOSY NMR spectra of pure HbPGL, poly(AM-ran-APBA) in
solution
All measurements were carried out at 295K on a Bruker Avance III 500 spectrometer
equipped with a 5 mm BBI probe head with zꢀgradients coil and GAB/2 gradient unit capable
of producing B gradients with a maximum strength of 50 G/cm. The BCUꢀ05 cooling unit,
0
managed by the BVT3300 variable temperature unit, was used for temperature control and
stabilization. The spectrometer was controlled with PC computer running under Windows 7
(64 bit) OS with the TopSpin 3.1 programme.
1
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1
least 10 minutes before data accumulation, and the H π/2 pulse length was checked and
adjusted carefully for each sample and temperature. The standard Bruker pulse programme
dstebpgp3s was selected for measurements using double stimulated echo for convection
compensation and LED (Longitudinal Eddy Current Delay) using bipolar gradient pulses for
diffusion and 3 spoil gradients. The shape of all gradient pulses was sinusoidal, the gradient
spoil pulse was 0.6 ms, the delay for gradient recovery was set at 0.2 ms, and the LED was set
at 5 ms and held constant in all experiments. The gradient pulse (small delta; δ/p30) was kept
constant throughout the whole series of temperature measurements, and the diffusion time
(big delta; ꢂ/D20) was changed to achieve the desired signal attenuation at maximum gradient
strength.
The DOSY experiments were run in pseudo 2D mode with gradients varied exponentially
from 5% up to 95%, typically in 16 steps, with 16 scans per step.
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Spectra were processed by TopSpin 3.1 software supplied by the spectrometer manufacturer.
The 1 Hz line broadening Lorenzian function was applied and each row was phased and
baselineꢀcorrected before executing the Fourier transformation in the F2 dimension. Diffusion
1
coefficient values, for resolved H signals were extracted from the T
1 2
/T analysis module of
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the TopSpin 3.1 programme.
NMR spectra of polymer solutions were recorded at deuterated PBS (pH=7.40).
- High Resolution Magic Angle Spinning NMR, HR MAS NMR
HR MAS NMR analyses of hydrogel samples were prepared in PBS buffer composed of
H
2
O:D
2
O mixture (9:1, v/v). The small additive of deuterium water was necessary for locking
and shimming the spectrometer to obtain spectra of high quality, which is particularly
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important for measurements conducted at different values of temperature.
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H Diffusion-Ordered Spectroscopy ( H DOSY)
All HR MAS NMR experiments were conducted on a Bruker Avance III 400 spectrometer,
with a Ultra Shielded Plus Wide Bore superconducting magnet, operating at 400.13 MHz for
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the H Larmor frequency. The spectrometer was equipped with a 4 mm triple resonance
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H/ C/ P HR MAS probe head with a deuterium ( H) lock channel and magic angle gradient
coil. The spectrometer was equipped with a GAB/2 gradient unit capable of producing B
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gradients with a maximum strength of 50 G/cm. Temperature control and stabilization was
achieved by controlling the bearing gas temperature with a BCU05 driven by the BVT3200
temperature unit. For MAS rotation and control, a MAS II unit (Bruker) was utilized. Both
acquisition and processing were conducted using the original computer (HPꢀ XW 4400
Workstation) and software (TopSpin 2.1 running under Windows XP Professional) supplied
by the manufacturer of the spectrometer.
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All HR MAS experiments were recorded using 4 mm zirconia rotors with KelꢀF caps. The gel
sample was directly placed and sealed in BL4 HRꢀMAS KelꢀF inserts (Bruker) and then were
placed in the rotor. For all measurements, the MAS spinning rate was set to 10 kHz, and
before running each experiment, the sample was kept at the desired temperature and rotation
for 15 minutes. The pulse sequence based on the simulated echo and incorporating bipolar
gradients pulses for diffusion with one spoil gradient and water suppression using a 3ꢀ9ꢀ19
pulse sequence with a gradient, was used without any changes from the Bruker pulse
programme library (stebpgp1s19). Before DOSY spectrum accumulation at each temperature,
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the probe head was tuned and matched carefully, and the H π/2 pulse length and offset (O1 ꢀ
for water suppression) were checked and corrected. The 1D spectra, both with and without
presaturation, were accumulated before DOSY measurement. Both the gradient pulse (small
delta; δ) and the diffusion time (big delta; ꢂ) were set for each temperature separately. The
gradient spoil pulse was 0.6 ms, and the delay for gradient recovery was set to 0.2 ms. The
DOSY experiments were run in pseudo 2D mode with 24 increments and 24 scans for each
increment. The shape of all gradient pulses was sinusoidal and the strength was changed
exponentially between 5 and 95 percent of the maximum value. The spectra were processed
using the TopSpin 2.1 software supplied by manufacturer of the spectrometer. The 1 Hz line
broadening Lorenzian function was applied and each row was phased and baselineꢀcorrected
before executing the Fourier transformation in the F2 dimension. The diffusion coefficients
1
for resolved H signals were extracted using T
1 2
/T analysis module of the TopSpin 2.1
programme.
The average values of diffusion coefficients of all components building the hydrogel, i.e.,
polymer components building the hydrogel and in situꢀgenerated lowꢀmolecularꢀweight
product, i.e., pꢀnitrophenol were determined based on the diffusion coefficient of proton
signals corresponding to appropriate components. For example, at 283 K, diffusion coefficient
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of HbPGL was estimated based on multiplet signal at δ=3.70 ppm, whereas the diffusion
coefficient of poly(AMꢀranꢀAPBA) was estimated based on protons in the aromatic region
(6.90ꢀ7.90 ppm) and corresponding to the main copolymer backbone (1.40ꢀ2.50 ppm). The
diffusion coefficient of pꢀnitrophenol was estimated based on signals of two dublets located
respectively at approximately 6.80 ppm and 8.20 ppm.
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P HR MAS NMR
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The biomineralization process was confirmed based on the P HR MAS NMR spectra. The
4K data points and 128 scans FIDs were accumulated using the original lowꢀpower
decoupling pulse sequence (zgpg) from the Bruker pulse programme library. The observe
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pulse ( P π/2) was set at 10 ꢁs, and the 2,5 kHz pulse was applied for H decoupling with the
garp sequence. The 10 Hz apodisation filter, without zeroꢀfilling, was applied prior to Fourier
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transformation. The chemical shift was referenced externally to the P shift of the 85%
H
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PO sample (0.00 ppm).
4
RESULTS AND DISCUSSION
1.
Formation of HbPGL/poly(AM-ran-APBA) hydrogels and their rheological
properties
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We have prepared several model hydrogel systems composed of hyperbranched
polyglycidol (M
MnCOP2=41000, each one bearing 7.4 mol % of 2ꢀacrylamidophenyl boronic acid units and
2.6 mol % of acrylamide (Scheme 2). Boronic acid groups react with 1,2ꢀdiol functional
n
=6400) crossꢀlinked with two acrylamide copolymers of MnCOP1=23000 or
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groups in the peripheral area of the macromolecule of HbPGL, generating the network by the
formation of boronic ester crossꢀlinks (Scheme 3). The detailed hydrogel composition is
presented in the Experimental Section. The gel formation based on boronic ester crossꢀlinks at
neutral pH in PBS buffer (pH=7.40) may be due to the intramolecular coordination between
the carbonyl oxygen of the acrylamide moiety of the copolymer and the boron atom in
[31]
boronic acid.
The coordination facilitates the tetrahedral form of boronic acid, which
preferably reacts with 1,2ꢀ or 1,3ꢀ diol groups in aqueous media. Our previous HbPGL
hydrogels crossꢀlinked with 1,4ꢀphenyl boronic diacid and borax were not suitable for
3
6-37
biological applications due to the gelation at basic pH.
Formation of the tetrahedral form
of boronic acid at neutral pH may be due to the introduction of boron coordinating atoms such
as nitrogen and oxygen, which have a lone electron pair, in the structure of the boronic acid
moietyꢀbearing molecule. Beside carbonylꢀcoordinating boronic acids, there can be also
distinguished heterocyclic analogues of boronic acids called as benzoxaboroles and Wulffꢀ
38
type boronic acid. In this article, we wanted to explore the influence of the acrylamide
copolymer length (gels H3A and H3B) on diffusion of gelꢀforming macromolecules as well as
on the mobility of entrapped molecules. Moreover, with the hydrogel H1, we wanted to
examine the influence of the weight fraction of polymers.
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Scheme 2. Polymer components applied for the formation of HbPGL/poly(AMꢀranꢀAPBA)
hydrogels.
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- hyperbranched polyglycidol
- poly(AM-ran-APBA)
polyacrylamide
site
CH2 CH
AP - alkaline phosphatase
C
N
O
B
-
denotes a cross-link
in the hydrogel
O
HbPGL site
O
boronic ester
cross-link
S P - substrate hydrolyzed to product
S = p-nitrophenyl phosphate or naphtyl phosphate calcium salt;
P = p-nitrophenol or calcium phosphate
Scheme 3. The structure of the hydrogel network composed of HbPGL crossꢀlinked with
poly(AMꢀranꢀAPBA) copolymer with incorporated alkaline phosphatase and in situꢀ
generated product of the hydrolysis of encapsulated organic phosphates.
Frequency sweep tests of all investigated hydrogels revealed that the storage modulus
does not cross the loss modulus at its maximum value (Figure 1A), which results from more
39
than one relaxation time . Therefore, we cannot directly determine the lifetime of boronic
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ester crossꢀlinkers in the network. This behaviour results from additional effect of physical
entanglements of poly(AMꢀranꢀAPBA) macromolecules, which have a higher concentration
c
in gels than its overlap concentration c*. However, we can compare the changes in ω as an
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indicator of any change in the association/dissociation reaction dynamic, for example, with a
variation in temperature (Figure 1B). The hydrogels presented herein are especially
susceptible to temperature variations because the diol – boronic acid crossꢀlinking reaction is
[33]
exothermic . Any change in temperature exerts an influence on the number of crossꢀlinks
between macromolecules. As a consequence, the macroscopic properties of the network are
affected. Frequency sweep tests’ results at various temperatures clearly demonstrate a
reduction of the crossꢀlink lifetime at higher temperatures (Figure 1B, Figures S6ꢀS8). The
effect is especially prominent in H3B gels consisting of poly(AMꢀranꢀAPBA) with a lower
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molecular weight, for which ω
c
shifted from 1.52 rad/s at 283 K to 8.40 rad/s at 310 K.
Concurrently, increased from 0.89 rad/ to 5.51 rad/s in the case of H3A. This finding
ω
c
indicates that copolymer entanglements play important roles in stabilizing hydrogels at an
elevated temperature when the association/dissociation reaction rate between diols and
boronic acid moieties increases.
The rheological measurements, i.e., frequency sweep tests, revealed that incorporation of
alkaline phosphatase into the HbPGL/poly(AMꢀranꢀAPBA) hydrogel systems results in small
changes in the continuity of the network, such as the crossover of the angular frequency, ω
c
,
where G’=G”, is shifted to a higher value in comparison to ω in the hydrogel without enzyme
c
(
Figure 1C). For example, when alkaline phosphatase was incorporated to H3A gel at a
concentration of 1.76 mg/ml, ω increased from 0.89 rad/s at 10°C to 1.58 rad/s with
reduction of the maximum plateau of the elasticity module. The higher amount of introduced
protein (3.52 mg/ml) caused a further ω shift to 1.91 rad/s, and consequently G’max(ω) was
c
c
further reduced. This behaviour can be explained by competing interactions of the enzyme
with boronic acid, as it is known that boronic acid is prone to reactions with aminoacids,
which possess an electron donating group for the boron atom such as serine, histidine, or
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lysine . In addition, these interactions can lead to irreversible enzyme inhibition.
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Figure 1. Frequency sweep tests recorded for the: pure hydrogels H1, H3A and H3B at 10 °C
H3 (A); hydrogel H3A_pure at 10, 20 and 37°C (B); hydrogel H3 with various concentrations
of alkaline phosphatase (0; 1.76 and 3.52 mg/ml) (C).
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Figure 2. The ω crossover as the function of temperature for various HbPGL/poly(AMꢀranꢀ
APBA) hydrogel systems containing alkaline phosphatase.
An important feature of HbPGL/poly (AMꢀranꢀAPBA) hydrogels is their selfꢀhealing
characteristics (SI, Figure S9), i.e., they repair without the need for any additional trigger and
exhibit identical mechanical properties. This behaviour results, among others, from the
reversible characteristics of crossꢀlinks. As revealed in the rheological studies, the rate of
continuous dissociation/reassociation processes is fast enough for the examined hydrogels to
exhibit selfꢀhealing properties even at room temperature. Another factor playing a key role in
the healing process is the mobility of polymer molecules, which facilitates interactions
42
between specific groups to repair the material.
As the interface between two pieces of hydrogels merges almost immediate, we
acknowledged that the dynamics of macromolecules in the hydrogel structure is sufficient and
that rapid crossꢀlinks reassociation is ensured. HbPGL macromolecules are conformally
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flexible , which surely facilitate reactive site recognition. However, no data about the
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transitional mobility of macromolecules, which also significantly affects the healing
properties of hydrogels, was available. To extract information about the mobility of
1
macromolecules in the hydrogel network, H HR MAS DOSY NMR measurements were
performed. With this technique, we were able to determine the average values of diffusion
coefficients corresponding to the polymer components making up the network. The
experimentally investigated selfꢀdiffusion of polymer associating networks has not been
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widely described, as previously noted by Tang et al.
The average state of macromolecules within the network is complex, as it is the result of the
continuous dissociation/reassociation of the crossꢀlink process, which is strictly dependent on
temperature. To estimate the state of polymer components in the hydrogel systems, we
recorded the diffusion coefficients of pure polymer components at the concentrations applied
for hydrogel formation. Pure polymer components move faster in comparison to their
behaviour in the hydrogel. The diffusion coefficient of pure HbPGL at 295 K increases from
ꢀ11
ꢀ11
2
2
.46 x10 to 7.60x10 m /s upon raising the temperature from 273 to 343 K. In the case of
both (AMꢀranꢀAPBA) copolymers, the diffusion coefficient hardly changed in the applied
ꢀ11
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temperature range and varied approximately 2.0x10 m /s.
HR MAS NMR experiments for all presented hydrogel systems revealed differences in the
mobility of HbPGL and acrylamide copolymer macromolecules within the network. HbPGL
species turned out to be faster diffusing polymer component and even lowering the
temperature to 10°C did not result in a comparable diffusion rate of both polymer
components. Still, the average state of HbPGL species moved faster in comparison to the
copolymer species. These results indicate that the network rearrangement relies mainly on the
movement of unbound HbPGL macromolecules rather than clusters of HbPGL associated
with the AMꢀAPBA copolymer. The compact structure of HbPGL in the form of the
h
nanospherical object (R =2.0 nm determined in dilute concentration based on the Stokesꢀ
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Einstein equation in D O at 295 K, C=1.25 wt%) makes it easier to diffuse in comparison to
2
more static macromolecules with a linear topology of poly(AMꢀranꢀAPBA) copolymers
h h
(COP1: R =9.38 nm; COP2: R = 6.84 nm determined in the diluted concentration, C=0.20
wt%), which, due to their significant length, in comparison to HbPGL, along with the number
of reactive groups, can be linked to several HbPGL molecules. In addition, they can be
engaged in physical chain entanglements within the network, as its concentration exceeds the
overlap concentration. The dynamics of the acrylamide copolymer is slowed down by multiꢀ
point interactions with HbPGL. Macromolecules of poly(AMꢀranꢀAPBA) are more stationary
in comparison to HbPGL, playing a role in the network skeleton. Moreover, due to the small
fraction of boronic acid applied for hydrogel formation, some HbPGL macromolecules may
be sterically repelled/hindered from reactive boronic acid groups by other, previously
incorporated HbPGL macromolecules.
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Figure 3. Diffusion coefficients of polymers in solution, i.e., HbPGL and poly(AMꢀranꢀ
APBA) and within hydrogel systems (at the same concentrations): A) H1, B) H3A, C) H3B,
presented as the function of the inverse of the temperature.
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The H HR MAS DOSY NMR results correspond well to the rheological data. An increase in
temperature loosens the hydrogel structure, as the lifetime of the crossꢀlinks is gradually
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reduced and the equilibrium of the reaction is shifted to substrates because the crossꢀlinking
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reaction is exothermic, and the macromolecules are more mobile in the network (Figure 3
A,B,C). In the H3B gel, for which the biggest shift in ω with temperature was observed, the
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greatest increase in macromolecules mobility was observed (Figure 3C). At 313K, both
polymer components in this gel almost reached the mobility observed in solution. In the case
of H1 and H3A gels, prepared with a higher molecular weight poly(AMꢀranꢀAPBA), the
influence of temperature is much less visible, especially when the mobility of poly(AMꢀranꢀ
APBA) is observed. However, the H1 gel, which contains a higher fraction of HbPGL
macromolecules (i.e., the higher molar fraction of diol groups per boronic acid group), is
slightly more sensitive to temperature (Figure 3A). We assume that at a higher diol to boronic
acid molar ratio in H1, the average momentary number of unbound macromolecules, which
do not have to overcome a dissociation energy barrier, is elevated.
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The composition of the hydrogel strictly determines its structure. The shift of ωcrossover to
higher values provides information about the shortage of crossꢀlinks lifetime. The exchange
reaction is faster, and the dynamics of the polymer macromolecules are higher. It is
noteworthy that robust molecular dynamics within the network should also exert an influence
on the diffusion of encapsulated compounds through the hydrogel.
2.
Enzyme activity in hydrogel systems
Since our main goal is the application of HbPGL/poly(AMꢀranꢀAPBA) hydrogel systems for
biomineralization, which is triggered by entrapped alkaline phosphatase, the issue of the
structural stability of the enzyme within the hydrogel matrix is of great importance. The
rheological data demonstrated some disturbance in the crossꢀlinking efficiency upon enzyme
addition, which could indicate some competing interactions, likely between boronic acid
moieties and functional groups of phosphatase. These interactions can result in structural
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changes in the active centre of the enzyme, making it inactive for catalysis of the hydrolysis
of organic phosphates. Garner has reported that the inhibition of lipase activity under the
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influence of phenylboronic acid occurs as a result of its reaction in trigonal form with serine
in the active centre of lipase. Although carbonylꢀcoordinated boronic acids are supposed to
occur in tetrahedral form at neutral pH, it is advisable to confirm whether phosphatase
remains active within HbPGL/poly(AMꢀranꢀAPBA) hydrogel because serine also plays a
significant role in the mechanism of organic phosphate hydrolysis catalysed by alkaline
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phosphatase .
We performed a simple experiment in which we compared the activity of the enzyme
in the native state to the one that was encapsulated for 24 hours within the gel and
subsequently released upon dilution in PBS buffer. The measured biological activity of the
released alkaline phosphatase was comparable to the activity of the enzyme in the native state.
This result confirmed that the HbPGL/poly(AMꢀranꢀAPBA) hydrogel environment has no
detrimental effect on the structure of the entrapped biomolecules. The neutral effect of
boronic acid present in our gels on the structure of the active centre of alkaline phosphatase is
most likely a result of the tetrahedral state of Oꢀcoordinated boronic acid, which preferentially
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reacts with the 1,2ꢀdiol functional groups of HbPGL extensively present in the hydrogel,
leading to the formation of thermodynamically favoured fiveꢀmembered cycles, rather than an
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interaction with the monohydroxyl group of serine .
Confirmation of the neutral influence of the network on the enzyme structure is
important. However, to fully confirm the utility of our systems for enzymatically triggered
biomineralization, evaluation of effective enzyme activity within the hydrogel was required.
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To achieve this goal, the MichaelisꢀMenten (K ) constant for each gel was determined and
compared with the K of phosphatase in solution. We prepared series of hydrogel systems of
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each hydrogel type enriched with different amounts of substrate, i.e., pꢀnitrophenyl phosphate
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with a constant concentration of alkaline phosphatase (1.76 mg/ml). Measurements of enzyme
activity were conducted at 37°C.
The appearance of pꢀnitrophenol, the product of hydrolysis of substrate catalysed by
alkaline phosphatase, revealed the enzyme activity within all hydrogel systems, confirming
that the active sites of encapsulated enzyme are stable in the hydrogel microenvironment and
there is sufficient diffusion of the substrate/product in the hydrogel systems. In Figure 4, the
effective alkaline phosphatase activity within hydrogel structures is compared with the
activity of the enzyme in the native state. A reduced effective enzyme activity was observed
within all hydrogel systems. The values of MichaelisꢀMenten constant determined for enzyme
entrapped in all hydrogel systems gradually increased from 5.76 for enzyme in solution up to
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9.65 mM for hydrogel H1 (Table 3). The maximum rate of reaction was lowest in the
hydrogel with the highest polymer fraction (H1) but highest in hydrogel H3B.
The most significant reduction of enzyme activity was observed within hydrogel H1,
which can be ascribed to the higher weight fraction (30%) of the polymers making up the
structure. Comparison of the enzyme activity within hydrogels composed of a 23% weight
fraction of polymer components (H3A and H3B) revealed enhanced enzyme inhibition in
hydrogel built from longer poly(AMꢀranꢀAPBA) (hydrogel H3A) . Since the molar ratio of
diol to boronic acid was identical, the change in enzyme activity resulted from physical
entanglements generated by the longer macromolecules of poly(AMꢀranꢀAPBA) causing a
more significant hindrance to diffusing molecules through the hydrogel.
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Figure 4. The effective biological activity of alkaline phosphatase entrapped within
HbPGL/poly(AMꢀranꢀAPBA) hydrogel systems compared with the activity of phosphatase in
the native form.
Table 3. The kinetic parameters describing the effective biological activity of alkaline
phosphatase in the native state and encapsulated in HbPGL/poly(AMꢀranꢀAPBA) hydrogel
systems.
Alkaline phosphatase
Native_1.76 mg/ml
H1_1.76 mg/ml AP
H3A_1.76 mg/ml AP
H3B_1.76 mg/ml AP
V
max, mM/min
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K , mM
1.33±0.06
0,30 ± 0,02
0.53±0.05
1.17±0.05
5.76±0.86
9,65 ± 1,70
8.94±1,12
7.23±2.88
Based on the comparison of K values of enzyme in pure form with enzyme entrapped in the
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hydrogel network, we can easily demonstrate the change of the affinity of enzyme towards the
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substrate. Generally, the increase in K of enzyme in hydrogel systems is evidence of reduced
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enzyme activity, which can be caused by structural changes in the enzyme introduced into the
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hydrogel structure, or by reduced accessibility of the substrate to the active site. Based on
the observation of a neutral impact of the polymer neighbourhood on the structure of active
centre of alkaline phosphatase, we ascribe the reduced effective biological activity of
phosphatase to the restriction of substrate/product diffusion generated by the crossꢀlinked
polymer components. This factor plays a decisive role in the biomineralization aspect of the
application. The released phosphate ions must encounter calcium ions and precipitate in the
form of calcium phosphate. If the diffusion of compounds is significantly reduced, the active
centres of the enzyme can be blocked and limit access of other phosphate ester molecules to
the active centre, causing a reduction of the effective biological activity of the enzyme and
efficiency of hydroxyapatite formation throughout the entire hydrogel.
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3.
Measurements of diffusion coefficients of p-nitrophenol within HbPGL/poly(AM-
ran-APBA) hydrogel systems
Due to diminished biological activity of alkaline phosphatase within hydrogel systems, which
we demonstrated was not a result of a loss of enzyme structural stability, we decided to
investigate the aspect of lowꢀmolecularꢀweight compound diffusion within HbPGL/poly(AMꢀ
ranꢀAPBA) hydrogel systems. We expected that the reduced enzyme activity could be
explained by the restricted transport of compounds flowing through the hydrogel. The values
of diffusion coefficients of lowꢀmolecular compounds in the hydrogel reflect, to some extent,
the rate of substrate approach to the active centre and the rate of product escape from the
active centre of the enzyme, providing space for other substrate molecules.
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