Diffusion-Controllable Biomineralization Conducted In Situ in

Sep 5, 2017 - At neutral pH, diol functional groups of HbPGL react with boronic acid of polyacrylamide to generate 3D network in water by the formatio...
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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 Downloaded from http://pubs.acs.org on September 6, 2017

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Diffusion-controllable biomineralization conducted in situ in hydrogels based on reversibly cross-linked hyperbranched polyglycidol

Mateusz Gosecki†, Slawomir Kazmierski †, Monika Gosecka†* † Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, ul. Sienkiewicza 112, 90-363 Lodz * [email protected]; 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 carbonylcoordinated 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 injectable, facilitating gap filing of bone tissue. The 1H HR MAS DOSY NMR studies reveal that acrylamide copolymer plays the role of the network framework, whereas HbPGL 1

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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 and calcium phosphate formation rate. The 1H HR MAS DOSY NMR is used to monitor diffusion low-molecular weight compound within hydrogels, whereas

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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 activity after extraction from their native environment.1 In addition to the storage aspect, it is also undertaken due to many specific applications,2 amongst which the catalysis of diverse chemical reactions is the most popular.

3,4,5,6

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 great importance and interest.7-8 It includes the usage of polymer hydrogel matrices enriched with enzymes for the biomineralization process. For this goal, alkaline phosphatase may be entrapped within the hydrogel structure,

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in which the enzyme triggers bone

biomineralization processes by catalysing the hydrolysis of organic phosphates 11. 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 biomineralization process were biopolymers such as chitosan,12 alginate,13 silk fibroin,14 and agarose,15 as well as synthetic polymers, i.e., poly(ethylene glycol),16 poly(vinyl alcohol),17 poly(vinyl pyrrolidone), 18 and poly(acrylic acid), 19 , 20 , 21 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 3

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hydrogel structure. However, the cross-linking reaction can have detrimental effects on the protein structure, including changes in the active centre. For example, in the case of alginate gelation with Ca2+ ions, the structure of the proteins can be changed due to competing interactions of the carboxyl groups.22 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 association-dissociation of cross-links. These properties can be triggered by a specific stimulus such as temperature23 and pH,24 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 cross-links. Here, we present a new type of hydrogel that is suitable for biomineralization applications, which is composed of hyperbranched polyglycidol (HbPGL) reversibly crosslinked with a copolymer of acrylamide and 2-acrylamidophenylboronic acid. Interest in the use of HbPGL in hydrogel formation results from its advantageous characteristics, such as biocompatibility,25 superhydrophilicity26 and antibiofouling/ protein repellent behaviours27-28. The topology of HbPGL and its flexibility facilitate the compartmentalization of various molecules within the macromolecule structure, i.e., drugs and labels, 29 among others. Moreover, the presence of 1,2-diol functional groups in the terminal units of HbPGL provides the possibility of the formation of cross-links with boronic acid in aqueous solutions and the creation of reversible, covalent hydrogel systems. Generally, the formation of diol-boronic acid cross-links is preferred at an alkaline pH.30 However, Deng et al reported the possibility 4

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of diol-boronic acid cross-links formation at acidic and neutral pH values31 when the boron atom is intramolecularly coordinated with an oxygen. Polyacrylamide is also a well-known hydrophilic and bioinert polymer.

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In addition copolymers acrylamide and 2-

acrylamidophenylboronic (poly(AM-ran-APBA)) acid can be easy synthesized.[31] The presence of a carbonyl oxygen atom close to the boronic acid moiety in 2acrylamidophenylboronic acid ensures the intramolecular coordination with the boron atom. In reversibly cross-linked polymer networks, dynamic events, i.e., the cross-link 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 crosslinking of the network. For example, in the case of networks based on diol-boronic acid crosslinks, it can be achieved by changing the temperature because crosslinking is an exothermic process 33 . 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(AM-ran-APBA) hydrogels with respect to reagents diffusion. We relate this dependence to the dynamics of hydrogels determined by rheological measurements and diffusion data. By using 1H 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 low-molecular weight probe – pnitrophenol. This molecule, which is the product of the hydrolysis of p-nitrophenyl phosphate, was also used to estimate the effective bioactivity of alkaline phosphatase within hydrogels. In addition, based on

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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 1-naphthyl phosphate calcium salt hydrolysis.

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EXPERIMENTAL SECTION Materials The p-nitrophenyl phosphate disodium salt hexahydrate and 1-naphthyl phosphate calcium salt trihydrate were used as received from Sigma-Aldrich. Acrylamide (Sigma-Aldrich) was recrystallized from acetone. Glycidol, 96%, was obtained from Sigma-Aldrich and distilled under a vacuum (61°C; 15 mmHg). Comonomer 2-acrylamidophenylboronic acid pinacol ester was prepared according to the procedure described in Ref.

[31]

The 2,2-

azobisisobutyronitrile, AIBN (Fluka) was recrystallized from ethanol. Alkaline phosphatase from bovine intestinal mucosa was purchased from Sigma-Aldrich. 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 from PBS composed of H2O and D2O (9:1, v/v). Synthesis of HbPGL, hyperbranched polyglycidol Hyperbranched polyglycidol was synthesized by anionic polymerization according to the procedure described by Sunder

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. Briefly, 10% of the hydroxyl groups in 1,1,1-

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 oil-bath 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 cation-exchange resin (Dowex 50 WX 4). The product was precipitated 6

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three times from methanol in acetone to remove cyclic by-products, dried under a vacuum and characterized by 1H NMR,

13

C NMR and GPC methods (chromatograms and NMR spectra

are shown in SI in Figures S1, S2 and S3, respectively).  = 1.56 and    = 6400;   / All experiments were carried out using HbPGL with  DB (degree of branching) = 0.61. The fraction of vicinal diol in the terminal units was estimated on the basis of the 13C 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 2-acrylamidophenylboronic 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 pinacol boronic esters and remove released pinacol. The exemplary 1H NMR spectra confirming the molar composition of copolymer are presented in SI in Figure S4 and Figure S5. Different molecular weights of poly(AM-ran-APBA) were obtained by changing the amount of introduced AIBN against the comonomers. For the preparation of a shorter copolymer, COP1 (i.e., MnCOP1= 23000, Mw/Mn=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 (Mw/Mn=2.16), it was 1000:1.

Preparation of HbPGL-co-/poly(AM-ran-APBA) hydrogel systems HbPGL/poly(AM-ran-APBA) 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

Molar ratio of Number of -B(OH)2 diol:boronic groups per 1 HbPGL acid macromolecule

H1

Weight fraction HbPGL of polymers, macromolecules wt% per macromolecules of poly(AM-ran-APBA) 30 15.2: 1 COP2

14.0:1

2.0

H3A

23

7.0: 1 COP2

6.5:1

4.4

H3B

23

4.0:1 COP1

6.5:1

4.4

Preparation of hydrogels for effective activity measurements of an alkaline phosphatase We have prepared a series of hydrogels differing in molar concentration of p-nitrophenyl 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 p-nitrophenyl phosphate, whereas the solution of poly(AM-ran-APBA) in PBS buffer 8

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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 Polymer hydrogels were prepared by dissolving each polymer in an independent stock solution. Polyglycerol was dissolved in the solution of p-nitrophenyl phosphate prepared in PBS buffer (pH=7.4), whereas the solution of poly(AM-ran-APBA) 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 p-nitrophenyl phosphate was hydrolysed to p-nitrophenol, which was used as a probe for small molecules diffusion using the model HbPGL/poly(AM-ran-APBA) hydrogels. The final concentration of p-nitrophenyl 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 0.2-µm 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(AM-ranAPBA), 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

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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. Hydrogel

Weight fraction polymers, wt%

of Concentration of naphthyl phosphate calcium salt, mg/ml 9.11

H1_biom*

30

H3A_biom*

23

9.11

H3B_biom*

23

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 plate-plate geometry of 8 mm diameter with a 0.3 mm gap on a Thermoscientific HAAKE MARS 40 rheometer. An exemplary measurement of self-healing 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 p-nitrophenyl phosphate as a substrate facilitated monitoring the efficient biological activity of the enzyme, by the release of p-nitrophenol (Scheme 1), which is a yellow-coloured product with the characteristic maximum absorbance at 405 nm. We have prepared series of each hydrogel 10

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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 p-nitrophenyl 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 p-nitrophenol 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 p-nitrophenol were carried using a Specord S 600 analytik jena instrument. Based on the change in absorbance, the Michaelis-Menten constant of the enzyme entrapped within the hydrogel, Km and the maximum rate of the reaction, Vmax. Km, Vmax were determined with the OriginPro non-linear 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 pnitrophenyl phosphate.

Scheme 1. Hydrolysis of p-nitrophenyl phosphate into p-nitrophenol catalysed by alkaline phosphatase.

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NMR analysis -

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H NMR and 1H 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 z-gradients coil and GAB/2 gradient unit capable of producing B0 gradients with a maximum strength of 50 G/cm. The BCU-05 cooling unit, 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. For 1H DOSY measurements, each sample was stabilized at the desired temperature for at least 10 minutes before data accumulation, and the 1H 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; ∆/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 baseline-corrected before executing the Fourier transformation in the F2 dimension. Diffusion coefficient values, for resolved 1H signals were extracted from the T1/T2 analysis module of 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 H2O:D2O 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 important for measurements conducted at different values of temperature.35

-

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H Diffusion-Ordered Spectroscopy (1H 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 the 1H Larmor frequency. The spectrometer was equipped with a 4 mm triple resonance 1

H/13C/31P HR MAS probe head with a deuterium (2H) lock channel and magic angle gradient

coil. The spectrometer was equipped with a GAB/2 gradient unit capable of producing B0 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 (HP- 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 Kel-F caps. The gel sample was directly placed and sealed in BL4 HR-MAS Kel-F 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 3-9-19 pulse sequence with a gradient, was used without any changes from the Bruker pulse programme library (stebpgp1s19). Before DOSY spectrum accumulation at each temperature, the probe head was tuned and matched carefully, and the 1H 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; ∆) 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 baseline-corrected before executing the Fourier transformation in the F2 dimension. The diffusion coefficients for resolved 1H signals were extracted using T1/T2 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 situ-generated low-molecular-weight product, i.e., p-nitrophenol 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(AM-ran-APBA) was estimated based on protons in the aromatic region (6.90-7.90 ppm) and corresponding to the main copolymer backbone (1.40-2.50 ppm). The diffusion coefficient of p-nitrophenol 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

The biomineralization process was confirmed based on the 31P HR MAS NMR spectra. The 64K data points and 128 scans FIDs were accumulated using the original low-power decoupling pulse sequence (zgpg) from the Bruker pulse programme library. The observe pulse (31P π/2) was set at 10 µs, and the 2,5 kHz pulse was applied for 1H decoupling with the garp sequence. The 10 Hz apodisation filter, without zero-filling, was applied prior to Fourier transformation. The chemical shift was referenced externally to the

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P shift of the 85%

H3PO4 sample (0.00 ppm).

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 (Mn=6400) cross-linked with two acrylamide copolymers of MnCOP1=23000 or MnCOP2=41000, each one bearing 7.4 mol % of 2-acrylamidophenyl boronic acid units and 92.6 mol % of acrylamide (Scheme 2). Boronic acid groups react with 1,2-diol functional groups in the peripheral area of the macromolecule of HbPGL, generating the network by the formation of boronic ester cross-links (Scheme 3). The detailed hydrogel composition is presented in the Experimental Section. The gel formation based on boronic ester cross-links 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 boronic acid.

[31]

The coordination facilitates the tetrahedral form of boronic acid, which

preferably reacts with 1,2- or 1,3- diol groups in aqueous media. Our previous HbPGL hydrogels cross-linked with 1,4-phenyl boronic diacid and borax were not suitable for biological applications due to the gelation at basic pH.36-37 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 moiety-bearing molecule. Beside carbonyl-coordinating boronic acids, there can be also distinguished heterocyclic analogues of boronic acids called as benzoxaboroles and Wulfftype boronic acid. 38 In this article, we wanted to explore the influence of the acrylamide copolymer length (gels H3A and H3B) on diffusion of gel-forming 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(AM-ran-APBA) hydrogels.

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S P

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S P

S P

AP

S P S P

S P

AP AP S P

S P S P

S P S P S P AP

S P

S P

S P

AP

S P

AP S P

AP S P S P

S P

- hyperbranched polyglycidol - poly(AM-ran-APBA) AP - alkaline phosphatase

polyacrylamide site

CH2

CH C

N

- denotes a cross-link in the hydrogel

O B

O O boronic ester cross-link

HbPGL site

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 cross-linked with poly(AM-ran-APBA) copolymer with incorporated alkaline phosphatase and in situgenerated 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 than one relaxation time39. Therefore, we cannot directly determine the lifetime of boronic ester cross-linkers in the network.40 This behaviour results from additional effect of physical entanglements of poly(AM-ran-APBA) macromolecules, which have a higher concentration in gels than its overlap concentration c*. However, we can compare the changes in ωc as an 18

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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 cross-linking reaction is exothermic

[33]

. Any change in temperature exerts an influence on the number of cross-links

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 cross-link lifetime at higher temperatures (Figure 1B, Figures S6-S8). The effect is especially prominent in H3B gels consisting of poly(AM-ran-APBA) with a lower molecular weight, for which ωc shifted from 1.52 rad/s at 283 K to 8.40 rad/s at 310 K. Concurrently, ωc increased from 0.89 rad/ to 5.51 rad/s in the case of H3A. This finding 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(AM-ran-APBA) hydrogel systems results in small changes in the continuity of the network, such as the crossover of the angular frequency, ωc, where G’=G”, is shifted to a higher value in comparison to ωc in the hydrogel without enzyme (Figure 1C). For example, when alkaline phosphatase was incorporated to H3A gel at a concentration of 1.76 mg/ml, ωc 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 ωc shift to 1.91 rad/s, and consequently G’max(ω) was 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 lysine41. In addition, these interactions can lead to irreversible enzyme inhibition. 19

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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(AM-ranAPBA) hydrogel systems containing alkaline phosphatase.

An important feature of HbPGL/poly (AM-ran-APBA) hydrogels is their self-healing 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 cross-links. As revealed in the rheological studies, the rate of continuous dissociation/reassociation processes is fast enough for the examined hydrogels to exhibit self-healing properties even at room temperature. Another factor playing a key role in the healing process is the mobility of polymer molecules, which facilitates interactions between specific groups to repair the material.42 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 cross-links reassociation is ensured. HbPGL macromolecules are conformally flexible 43 , 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 macromolecules in the hydrogel network, 1H 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 self-diffusion of polymer associating networks has not been widely described, as previously noted by Tang et al.44 The average state of macromolecules within the network is complex, as it is the result of the continuous dissociation/reassociation of the cross-link 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 2.46 x10-11 to 7.60x10-11 m2/s upon raising the temperature from 273 to 343 K. In the case of both (AM-ran-APBA) copolymers, the diffusion coefficient hardly changed in the applied temperature range and varied approximately 2.0x10-11 m2/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 AM-APBA copolymer. The compact structure of HbPGL in the form of the nanospherical object (Rh=2.0 nm determined in dilute concentration based on the Stokes22

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Einstein equation in D2O at 295 K, C=1.25 wt%) makes it easier to diffuse in comparison to more static macromolecules with a linear topology of poly(AM-ran-APBA) copolymers (COP1: Rh=9.38 nm; COP2: Rh = 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 multipoint interactions with HbPGL. Macromolecules of poly(AM-ran-APBA) 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(AM-ranAPBA) and within hydrogel systems (at the same concentrations): A) H1, B) H3A, C) H3B, presented as the function of the inverse of the temperature.

The 1H HR MAS DOSY NMR results correspond well to the rheological data. An increase in temperature loosens the hydrogel structure, as the lifetime of the cross-links is gradually 24

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reduced and the equilibrium of the reaction is shifted to substrates because the cross-linking reaction is exothermic,33 and the macromolecules are more mobile in the network (Figure 3 A,B,C). In the H3B gel, for which the biggest shift in ωc with temperature was observed, the 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(AM-ran-APBA), the influence of temperature is much less visible, especially when the mobility of poly(AM-ranAPBA) 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. The composition of the hydrogel strictly determines its structure. The shift of ωcrossover to higher values provides information about the shortage of cross-links 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(AM-ran-APBA) 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 cross-linking 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 25

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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 influence of phenylboronic acid 45 occurs as a result of its reaction in trigonal form with serine in the active centre of lipase. Although carbonyl-coordinated boronic acids are supposed to occur in tetrahedral form at neutral pH, it is advisable to confirm whether phosphatase remains active within HbPGL/poly(AM-ran-APBA) hydrogel because serine also plays a significant role in the mechanism of organic phosphate hydrolysis catalysed by alkaline phosphatase 46. 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(AM-ran-APBA) 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 O-coordinated boronic acid, which preferentially reacts with the 1,2-diol functional groups 47 of HbPGL extensively present in the hydrogel, leading to the formation of thermodynamically favoured five-membered cycles, rather than an interaction with the monohydroxyl group of serine47. 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. To achieve this goal, the Michaelis-Menten (KM) constant for each gel was determined and compared with the KM of phosphatase in solution. We prepared series of hydrogel systems of each hydrogel type enriched with different amounts of substrate, i.e., p-nitrophenyl phosphate 26

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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 p-nitrophenol, 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 Michaelis-Menten constant determined for enzyme entrapped in all hydrogel systems gradually increased from 5.76 for enzyme in solution up to 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(AM-ran-APBA) (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(AM-ran-APBA) 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(AM-ran-APBA) 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(AM-ran-APBA) hydrogel systems. Alkaline phosphatase

Vmax, mM/min

KM, mM

Native_1.76 mg/ml

1.33±0.06

5.76±0.86

H1_1.76 mg/ml AP

0,30 ± 0,02

9,65 ± 1,70

H3A_1.76 mg/ml AP

0.53±0.05

8.94±1,12

H3B_1.76 mg/ml AP

1.17±0.05

7.23±2.88

Based on the comparison of KM values of enzyme in pure form with enzyme entrapped in the hydrogel network, we can easily demonstrate the change of the affinity of enzyme towards the substrate. Generally, the increase in KM of enzyme in hydrogel systems is evidence of reduced 28

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enzyme activity, which can be caused by structural changes in the enzyme introduced into the hydrogel structure, or by reduced accessibility of the substrate to the active site.48 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 cross-linked 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.

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 low-molecular-weight compound diffusion within HbPGL/poly(AMran-APBA) 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 low-molecular 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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Unless any specific interactions between gel-forming polymers and encapsulated molecules occur, the diffusion rate of the farther is strictly dependent on the average dimension of the mesh size of the investigated hydrogel. It is well-known that the diffusion of low-molecular compounds can be reduced by the steric hindrance generated by polymer macromolecules, because the molecules can simply encounter the macromolecules during their movement. In addition, cross-linking density can significantly modify the hydrogel diffusivity. 49,50,51 As a probe, we used p-nitrophenol, which was generated in situ within the hydrogel as a result of the alkaline phosphatase-catalysed hydrolysis of the incorporated p-nitrophenyl phosphate. Within all three HbPGL/poly(AM-ran-PBA) hydrogels, a constant amount of p-nitrophenyl phosphate was introduced. In approximately 15 minutes after sample preparation in all investigated hydrogels, the entire amount of phosphate was hydrolysed, which was confirmed by HR MAS 1H NMR. Then, 1H HR MAS DOSY NMR measurements were performed to determine the diffusion coefficient of p-nitrophenol inside the hydrogel. In Figure 5A, we present the temperature dependence of the diffusion coefficients of pnitrophenol within various HbPGL/poly(AM-ran-APBA) hydrogel systems. Generally, the diffusion coefficient of p-nitrophenol in each of the examined gel is lower than that in solution. We observed an increasing trend in the diffusion coefficient values of p-nitrophenol with a rising temperature from 280 to 350 K, but we can observe different slopes of that dependence for each hydrogel systems.

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Figure 5. The dependence of the p-nitrophenol diffusion coefficient in HbPGL/poly(AM-ranAPBA) hydrogels on temperature ranging from 280 to 350 K (A) and the diffusion quotient of p-nitrophenol determined in the temperature range from 293 to 323 K (B).

To eliminate the temperature effect on p-nitrophenol intrinsic mobility, we determined the diffusion quotient of p-nitrophenol within all investigated hydrogels (Figure 5B). The diffusion quotient, also called the hindrance factor,52- 53 is a ratio of the diffusion coefficient of p-nitrophenol in the network to its diffusion coefficient in the solution (at the same concentration). This approach demonstrates the real restriction of the diffusivity of the introduced probe within the hydrogel generated by the polymer components making up the 3D network. As the formation of boronic ester cross-links is exothermic and diffusional measurements revealed the enhanced movement of polymers making up the network at elevated temperature, we expected that upon increases in temperature, the mesh size of the hydrogel would generally increase and the movement of the substrate/product would accelerate. The dependence of diffusion quotient on temperature for H1, H3A and H3B hydrogels varies significantly. For the hydrogel composed of 30 wt% of polymer content (H1_1.76 mg/ml AP), the values of the diffusion quotient were smallest amongst all hydrogels. Moreover, the value

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was almost constant (ca. 0.18) in the whole range of temperature. The steric hindrance generated by the polymer content is probably so significant that the value of the diffusion quotient does not depend on the cross-linking density. In the case of hydrogels composed of 23 wt % of polymer content, the trend observed for the diffusion quotient on temperature was different. For hydrogel H3A_1.76 mg/ml AP generated from the acrylamide copolymer, Mn=41000, raising the temperature from 293 K to 323 K caused a slight decrease in the pnitrophenol diffusion quotient. In this system, the network is additionally constrained by entanglements of the long acrylamide copolymer. Additionally, an enhanced exchange reaction between macromolecules at higher temperature makes the network less defined, but still coherent, than at a lower temperature. This phenomenon probably causes some restrictions to the flow of molecules through the quickly rearranging hydrogel network. Conversely, a significant increase in p-nitrophenol diffusivity through the hydrogel H3B composed of the shorter poly-(AM-ran-APBA), Mn=23000, was observed with a rising temperature. This trend is consistent with the rheological measurements, showing shorter relaxation times (i.e., higher values of ωcrossover) in H3B than in H3A hydrogels. The H3B hydrogel network is looser because the crossover of the angular frequency is shifted to higher values in the investigated temperature range. As the molar fraction of the comonomer bearing boronic acid is similar in both copolymers, this behaviour can be ascribed to a reduced degree of physical entanglement, without which the network loses its integrity at higher temperatures, enabling faster p-nitrophenol diffusion. Generally, diffusion data clearly explain the direction of phosphatase activity reduction within hydrogel systems. As we knew the order value of the diffusion coefficients of low-molecular compound within the hydrogel, we decided to estimate the relationship between the diffusion rate of substrate incorporated into the hydrogel and the possible diffusion of alkaline phosphatase (molecular weight approximately 185 kDa54 ). However, an insufficient amount of enzyme 32

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entrapped in the hydrogel made it impossible to monitor the enzyme diffusion by 1H HR MAS DOSY NMR. We elaborated, however, a simple experiment to visualize diffusion within the gel by taking advantage of the nearly colourless solution of p-nitrophenol phosphate and intense yellow solution of p-nitrophenol. In a Teflon vial covered with polycarbonate plate, a sample made of three hydrogels was prepared, i.e., the first contained p-nitrophenyl phosphate (on the left), the second – pure hydrogel (in the middle), and last one had entrapped alkaline phosphatase (on the right), (Figure 6). The barriers between them immediately disappeared due to the rapid self-healing properties of the applied hydrogels. The entire length of all hydrogels was 3.0 cm, and the entire volume of a sample was 0.45 cm3.

This experiment demonstrated that in approximately 24 hours, p-nitrophenyl phosphate (substrate) diffused across the pure hydrogel to the hydrogel block with entrapped alkaline phosphate, as indicated by the appearance of yellow colour at the verge of both gels. Afterwards, the whole sample gradually became yellow due to p-nitrophenol diffusion. The experiment clearly showed that the diffusion rate of alkaline phosphatase was meaningless in comparison to p-nitrophenol phosphate.

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Figure 6. Experiment demonstrating p-nitrophenyl phosphate migration through the hydrogel from left to the right along the neat hydrogel and release of p-nitrophenol (yellowish colour). In a Teflon container in one line, three types of hydrogels were placed: hydrogel containing p-nitrophenyl phosphate (on the left), in the middle – the neat hydrogel, and on the right, the hydrogel with incorporated alkaline phosphatase. Curves on each frame denotes an approximate p-nitrophenyl phosphate (substrate) concentration profile across the middle section of the hydrogel sample. Profiles were determined from the Fick’s second law and are scaled, as follows the top of each hydrogel frame corresponds to the initial substrate concentrations (in the left part of the hydrogel sample), whereas its bottom part correspond to 34

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zero concentration of p-nitrophenyl phosphate. As yellow colour appears on the border between the neat hydrogel and that containing the enzyme, we establish that the migration of enzyme through the network, in comparison to the transport rate of substrate/product, is negligible. 4.

Biomineralization in the HbPGL/poly(AM-ran-APBA) hydrogel system For efficient biomineralization to occur in hydrogel material, i.e., calcium phosphate

(CaP) formation in situ upon catalysis of alkaline phosphatase, two main factors are very important. In addition to the structural stability of alkaline phosphatase in the environment of the hydrogel, sufficient diffusion of all reaction components, i.e., substrates and products, is required because AP biomolecules are sparsely localized in the hydrogel network. Thus, molecules of phosphate substrate must diffuse to the active centre of AP and undergo hydrolysis into product, which must diffuse away to facilitate the approach of the next substrate molecule to the active centre. Experiments with p-nitrophenol have previously shown that the rate of hydrolysis in studied hydrogels is diminished with respect to the reaction carried out in solution. However, we expected that the rate of naphthyl phosphate hydrolysis would be even slower because precipitated calcium phosphate may cause additional hindrance to the diffusion of reactants. The progress of biomineralization within hydrogels was investigated with 31P HR MAS NMR spectroscopy (Figure 7). The concentration of alkaline phosphatase and naphthyl phosphate calcium salt, in all hydrogel samples were 1.76 mg/ml and 9.11 mg/ml, respectively. 31P HR MAS NMR spectroscopy confirmed the formation of calcium phosphate (chemical shift, δ=0.60 ppm, see SI, Figure S10-S12). The time needed for complete conversion of introduced organic phosphate within H1_biom and H3A_biom hydrogels was approximately 5 hours and 3.5 hours, respectively. In the case of H3B gel, the time of

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hydrolysis of introduced organic phosphate was substantially shorter, at less than 110 minutes. This trend is consistent with the diffusion coefficient values of p-nitrophenol and effective biological activity of phosphatase determined within those hydrogels. In fact, the hydrolysis of naphthyl phosphate calcium salt was longer in comparison to the hydrolysis of p-nitrophenyl phosphate. This behaviour can be explained by steric hindrance generated by precipitated solid CaP, which is insoluble in the hydrogel matrix, in contrast to water soluble p-nitrophenol, creating a diffusion barrier to the approach of other ester molecules to the enzyme active centre. The

31

P HR MAS NMR spectra recorded for all

hydrogel samples demonstrated that, from the beginning of the reaction, one very narrow signal at δ =0.60 ppm corresponded to calcium phosphate. According to the literature, a linewidth of signal in the

31

P magic angle spinning (MAS NMR) data below 0.63 ppm is

typical for crystalline phase of calcium phosphate. 55-56 The

31

P HR MAS NMR spectra of the

hydrogels did not change after one week. It is noteworthy that up to one hour, all hydrogel samples were transparent. Calcium phosphate precipitation within H3A_biom and H3B_biom hydrogels was visible with the naked eye. H3A_biom and H3B_biom hydrogels started to be opaque after around two hours of their formation, whereas even after one week, hydrogel H1 was still transparent. SEM images revealed distinct differences in the morphology of hydrogel systems upon biomineralization (Figure 8 and Figures in SI.7-SI.9 parts). Within the structure of H1_biom hydrogel, we observed a slight decrease in the pore diameter, which results from the network saturation with CaP. There were also numerous, isolated precipitated CaP objects. Significant diffusion restriction probably leads to a local accumulation of released phosphates, which upon reaction with calcium ions form calcium phosphate particles of approximetely 150 nm only. SEM images of the hydrogel H3A_biom revealed only a small change in the polymer

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material network, and no pronounced saturation of the network with visible CaP was observed apart from small structures occurring at the edges of the network. Most importantly, however, there were also several sparsely distributed several and more micron-sized CaP crystals, which probably resulted from the local supersaturation leading to the formation of CaP crystal seed in these regions rather than evenly throughout the whole volume of gel 57. The growth of well-defined crystals results from the proper balance between the hydrolysis rate, which determines the CaP concentration in the gel, and the diffusion of the components through the network, which ensures a relatively low supersaturation of the sample. In the case of hydrogel H3B_biom, the SEM images did not reveal any separated CaP objects. However, the most significant reduction in voids size was noticed, indicating that CaP was evenly distributed within the polymer network in the form of small, indistinguishable SEM image particles. Nevertheless, NMR experiments showed that formed CaP was present only in crystalline form only. The formation of small crystals is probably the result of a relatively rapid substrate conversion, which upon relatively small diffusion restrictions quickly leads to supersaturation of the whole sample with CaP. SEM studies of biomineralized hydrogels revealed that, in fact, small changes in the composition of hydrogels, such us the molecular weight of one of the polymer components, has a profound influence on the morphology of the formed CaP. However, we must remember that crystal growth is only the last step in the series of interconnected processes leading ultimately to biomineralization. As described previously, the rate of diffusion of reactants plays an important role in all these steps, ultimately defining the morphology CaP. Of course, we realize that the diffusion of molecules within the hydrogel depends on the network structure and the occurrence of any specific interaction between the gel and the entrapped molecules. In a dynamically cross-linked network, however, we can explicitly define its structure only in the time frame shorter than the lifetime of the cross-links. From this 37

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perspective, a more important role is played by the dynamics of the network, which is embodied both by its rheological properties and the diffusion of gel-forming polymers. Nevertheless, our approach to the biomineralization process conducted within hydrogel systems, facilitates the formation of insoluble calcium phosphate homogeneously in the whole bulk of the hydrogel. In contrast, the biomineralization process relies on hydrogel immersion in solution enriched with phosphate ester, which leads to biomineralization of the hydrogel structure mostly in its outer region. 58

Figure 7. Conversion of 1-naphthyl phosphate calcium salt within HbPGL/poly(AM-ranAPBA) hydrogels.

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Figure 8. SEM images of neat HbPGL/poly(AM-ran-APBA) hydrogels and hydrogels with biomineralization triggered in situ by alkaline phosphatase.

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CONCLUSIONS We have demonstrated that self-healing, injectable hydrogels based on HbPGL and poly(AM-ran-APBA) can be effectively used as a matrix for enzymatic biomineralization. The system utilizes a reversible reaction between phenyl boronic acid and 1,2-diol peripheral groups of HbPGL, which occurs at neutral pH. We observed that the hydrogel microenvironment is neutral to the active centres of the enzyme. In the presented hydrogel systems, the decrease in enzyme activity could be directly related to the reduced diffusion of encapsulated molecules through the hydrogel polymer matrix, as good mobility of both substrate and product molecules is required for efficient enzymatic reaction. Additionally, the results supported a direct relationship between the mobility of encapsulated molecules and the network dynamics.

Due to the gelation under neutral conditions in the presented system and neutral influence of the hydrogel environment on the enzyme, our hydrogel system is suitable for studying the relationship between the network structure, transport rate of low-molecularweight compounds and efficiency of enzymatic processes that occur within the hydrogel. As the presented system is fully synthetic, its properties can be easily modified to meet certain demands. We have shown that simply by modifying the length of the cross-linker, we changed the network consistency, which affected diffusion through the network and ultimately the morphology of the formed calcium phosphate crystals. This feature is highly important from the perspective of the mechanical properties of repaired bone. It is noteworthy that the reversible characteristics of cross-linking and sufficiently low molecular weight of

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polymer building components ensures the ultimate removal of all polymer components from an organism making the HbPGL-poly(AM-ran-APBA) hydrogel an attractive system. The morphology of in situ-formed calcium phosphate crystals within hydrogel systems is strictly correlated with the diffusion of substrates and products, which affects the process at defferent steps (enzymatic hydrolysis, crystallization). To better understand all correlations within this process, we plan to further investigate the presented systems, which would allow the design of a system that fulfils any particular medical requirement.

ASSOCIATED CONTENT The Supporting Information includes: 1H, 13C NMR spectra and GPC analyses of hyperbranched polyglycidol and poly(AM-ran-APBA), rheological measurements of hydrogels and SEM images of biomineralized hydrogels with 31P HR MAS NMR spectra.

ACKNOWLEDGEMENTS We are very grateful to D. Sc. Grzegorz Lapienis for performing the GPC analyses. Funding Sources Presented research was supported by the National Science Centre, Poland (project number: UMO-2015/17/D/ST5/02458).

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Figure 1. Frequency sweep tests recorded for: 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). 63x48mm (300 x 300 DPI)

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Figure 2. ω crossover in the function of temperature for various HbPGL/poly(AM-ran-APBA) hydrogel systems containing alkaline phosphatase. 59x41mm (300 x 300 DPI)

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Figure 3. Diffusion coefficients of polymers in solution, i.e., HbPGL and poly(AM-ran-APBA) and within hydrogel systems (at the same concentrations) A) H1, B) H3A, C) H3B presented in the function of the inverse of temperature. 176x370mm (300 x 300 DPI)

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Figure 4. The effective biological activity of alkaline phosphatase entrapped within HbPGL/poly(AM-ranAPBA) hydrogel systems compared with the activity of phosphatase in the native form. 67x54mm (300 x 300 DPI)

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Figure 5. The dependence of p-nitrophenol diffusion coefficient in HbPGL/poly(AM-ran-APBA) hydrogels on temperature in the range from 280 to 350 K (A) and diffusion quotient of p-nitrophenol determined in the temperature range from 293 to 323 K (B). 32x12mm (300 x 300 DPI)

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Figure 6. Experiment demonstrating p-nitrophenyl phosphate migration through the hydrogel from left to the right along the neat hydrogel and release of p-nitrophenol (yellowish colour). In a Teflon container in one line, three types of hydrogels were placed: hydrogel containing p-nitrophenyl phosphate (on the left), in the middle – the neat hydrogel, and on the right, the hydrogel with incorporated alkaline phosphatase. Curves on each frame denotes an approximate p-nitrophenyl phosphate (substrate) concentration profile across the middle section of the hydrogel sample. Profiles were determined from the Fick’s second law and are scaled, as follows the top of each hydrogel frame corresponds to the initial substrate concentrations (in the left part of the hydrogel sample), whereas its bottom part correspond to zero concentration of pnitrophenyl phosphate. As yellow colour appears on the border between the neat hydrogel and that containing the enzyme, we establish that the migration of enzyme through the network, in comparison to the transport rate of substrate/product, is negligible. 37x38mm (220 x 220 DPI)

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Figure 6. Experiment demonstrating p-nitrophenyl phosphate migration through the hydrogel from the left to the right along the neat hydrogel and release of p-nitrophenol (yellowish color). In Teflon container, in one line, three types of hydrogels were placed: hydrogel containing p-nitrophenyl phosphate (on the left), in the middle – the neat hydrogel, and on the right, the hydrogel with incorporated alkaline phosphatase. 67x53mm (300 x 300 DPI)

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Figure 8. SEM images of neat HbPGL/poly(AM-ran-APBA) hydrogels and those hydrogels upon biomineralization triggered in situ by alkaline phosphatase. 174x197mm (300 x 300 DPI)

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Scheme 1. Hydrolysis of p-nitrophenyl phosphate into p-nitrophenol catalyzed by alkaline phosphatase. 30x6mm (300 x 300 DPI)

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Scheme 2. Polymer components applied for the formation of HbPGL/poly(AM-ran-APBA) hydrogels. 129x92mm (300 x 300 DPI)

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Scheme 3. The structure of hydrogel network composed of HbPGL cross-linked with poly(AM-ran-APBA) copolymer with incorporated alkaline phosphatase and in situ generated product of hydrolysis of encapsulated organic phosphates. 144x131mm (300 x 300 DPI)

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