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Local environment-dependent kinetics of ester hydrolysis revealed by direct 1H NMR measurement of degrading hydrogels
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Local environment-dependent kinetics of ester hydrolysis revealed by direct 1 H NMR measurement of degrading hydrogels Chi Ming Laurence Lau a,b, Ghodsiehsadat Jahanmir a,b, Ying Chau a,b,∗ a b
The Hong Kong University of Science and Technology Shenzhen Research Institute, Shenzhen, China Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong, China
a r t i c l e
i n f o
Article history: Received 31 May 2019 Revised 5 October 2019 Accepted 23 October 2019 Available online xxx Keywords: Hydrogel Ester hydrolysis Degradable NMR Dextran Peg Hyaluronic acid
a b s t r a c t We have demonstrated the use of a simple 1 H NMR spectrometry-based method to directly measure the pseudo first-order hydrolytic cleavage rate constant (kobs ) of methacrylate-derived ester crosslinkers in hydrogels composed of PEG, dextran, carboxymethyl dextran (CM-dextran) and hyaluronic acid (HA). Using this technique, we systematically examined how the local environment in the hydrogel influenced the rate of ester hydrolysis. Within the formulations being studied, the esters in the crosslinked polymer network (gel state) degraded 1.8 times faster than esters of similar chemistry in soluble polymers (solution state). Furthermore, the value of kobs was independent of the polymer concentration or the hydrogel network structure, although these parameters affected the swelling profiles in response to the hydrolytic degradation. On the other hand, the presence of the negatively charged carboxylate groups in the polymer chains decreased kobs in gel state, while only minimally affecting kobs in solution state. Hydrogels composed of negatively charged polymers (HA and CM-dextran) had a kobs about 30% smaller than hydrogels composed of neutral polymers (dextran and PEG). The reported method provides a reliable tool to resolve conflicting views about hydrogel degradation, and to guide the rational design of degradable hydrogel. Statement of significance Degradable hydrogels are widely used in biological applications. A common degradation mechanism of the crosslinked polymer is by hydrolytic cleavage. However, the hydrogel micro-milieu do affect the behavior of the hydrolysable bonds, for example esters. There have been several conflicting speculations on how hydrogel composition would affect the macroscopic degradation behavior. In this report, we simply, but innovatively applied ordinary 1 H NMR spectrometry-based method to probe the rate of ester cleavage in the native hydrogel milieu. We tried to answer whether these parameters will have direct influence on ester cleavage, or have indirect effect on the overall network disintegration behavior. This study provides quantitative evidences to assist theoretical modeling and to guide rational formulation design. © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction A hydrogel is a water-retaining network composed of hydrophilic polymers in an aqueous environment. The unique properties of a hydrogel have attracted intensive research about its potential applications in biomedical areas [1–4]. For many applications, a predictable and controllable degradation profile is advantageous. Formation of the hydrogel network is via chemical or physical crosslinking between precursor polymers, and/or ∗ Corresponding author at: The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China. E-mail address: [email protected] (Y. Chau).
monomers, and hydrogel degradation can be achieved by hydrolysis [5–10], enzymatic digestion [11,12], or photolysis [13,14] of the polymer network. Commonly utilized hydrolysable moieties include anhydrides, esters and amides, among which esters have a moderate hydrolytic half-life (at the scale of days) under physiological conditions. Since the polymer network in hydrogel is highly hydrated, bulk degradation dominates the erosion of most hydrolytically degradable hydrogels [15]. Thorough understanding of the influence of hydrogel parameters on the macroscopic degradation profile would be extremely helpful to guide the rational formulation design. However, these parameters are usually interdependent, making the process of bulk degradation too complex to be described using simple equations. Theoretical modelling
https://doi.org/10.1016/j.actbio.2019.10.030 1742-7061/© 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: C.M.L. Lau, G. Jahanmir and Y. Chau, Local environment-dependent kinetics of ester hydrolysis revealed by direct 1 H NMR measurement of degrading hydrogels, Acta Biomaterialia, https://doi.org/10.1016/j.actbio.2019.10.030
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approaches, such as the mass loss model [16,17] and equilibrium swelling-model [9] are widely used to describe and predict the macroscopic degradation phenomena of hydrolysable hydrogels quantitatively [18–23]. In general, the models adopt a two-step approach: construct a numeric polymer network first, then simulate the process of network disintegration and calculate the corresponding output parameters. In the first step, the initial properties of the polymer network, such as the crosslinking density and distribution of degradable bonds in the network, are calculated based on the linker functionality (number of crosslinkers per chain) and polymer concentration (the number of polymer chains). Polymer crosslinking is often assumed ideal, although certain more sophisticated models also include non-idealities in the algorithm [18–21]. In the second step, a rate of network dissociation is assigned to the cleavable ester linkages in hydrolysable hydrogels, and models will generate measurable parameters such as polymer mass loss, and/or gel swelling ratios as outputs. The hydrolytic cleavage kinetics is described using a pseudo first-order rate constant (kobs ). The kobs is a lumped parameter that includes the external environmental parameters, such as temperature, pH, ionic strength etc. The values of ester kobs are usually indirectly measured by HPLC or NMR based methods in soluble hydrogel precursors, and generalized to gel state at which the precursors are crosslinked. In limited cases, kobs in hydrogel can be directly determined by following the release profile of ester-conjugated moieties from the hydrogel [23]. However, assuming an equal kobs value before and after gelation lacks experimental evidences. There have been some conflicting speculations about whether the kobs of an ester is the same in hydrogel formulations that differ in degree of network crosslinking [24], polymer concentration [21,25], and the network charge [23]. Several studies have reported that hydrogel degradation behavior varied significantly in different formulations, although the esters shared the same chemistry [9,16,25,26]. The observations indicate that kobs may be hydrogel milieu-dependent. However, it is also possible that the swelling profile and disintegration time can be significantly changed by altering polymer concentration only, with the kobs remaining unchanged [21]. On the other hand, the value of kobs is often assumed to be constant through the degradation process if the external environmental parameters do not change, which may not be true. Since the hydrogel structural parameters are changing in a degrading hydrogel, we anticipate that kobs will change during hydrogel degradation. Therefore, a technique to determine the kobs in the specific hydrogel environment is valuable to resolve the conflicting views about hydrogel degradation. Here, we adopt a simple method using conventional 1 H NMR spectroscopy to directly measure the kobs of the ester crosslinkers in hydrogels. The 1 H NMR has been routinely used in hydrolysis rate measurement for different materials in solution state [16,27]. For certain ester species, the signature shift of carbonyl neighboring protons can be easily differentiated before and after hydrolysis. The hydrolysis kinetics can be followed by tracking the rate of consumption of intact esters, or the generation of hydrolyzed esters. In this study, we have extended the method to the gel state, in which the polymers are covalently crosslinked. To demonstrate the utility of this approach, hydrolytically degradable hydrogels were formulated using various common polymers, including polyethylene glycol (PEG), dextran and hyaluronic acid (HA). The values of kobs were determined for formulations varying in polymer concentration, polymer conformation and crosslinker functionality, as well as the network charge. Since the method allows direct measurement of kobs in gel state, the contribution of the hydrogel’s local environment can be systematically studied.
2. Material and methods 2.1. Synthesis of vinyl sulfone functionalized dextran (DX40k-VS), carboxymethyl-dextran (CMDX40k-VS) and hyaluronic acid (HA29k-VS) The 8-arm PEG20k-VS (95%) was purchased from JenKem Technology, USA. Carboxymethyl-dextran-40k (CMDX-40k) was prepared based on a previously reported method [28]. Vinyl sulfone (VS)-functionalized hydroxyl-rich polysaccharides were synthesized as previously reported [29]. In brief, polymers were dissolved in dilute sodium hydroxide solution at desired concentrations. Divinyl sulfone (97% contains < 650 ppm hydroquinone as inhibitor, Aldrich) was added in excess (Scheme 1). The degree of VS conjugation was controlled by reaction time, and the reaction was stopped by pH decrease (
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Scheme 1. Conjugation of vinyl sulfone (VS) to hydroxyl rich polysaccharides.
Scheme 2. Conjugation of thiol group to dextran via ester linkers.
NaH2 PO4 and Na2 HPO4 stock solutions at different ratios (details are provided in the Table S5 in supplementary information). The apparent pH values were measured using an Orion micro pH electrode with Star A111 benchtop pH meter (Thermo-scientific). The value of pD can be quickly approximated by adding an empirical conversion equation [31]:
pD = pHa + 0.4 The phosphate buffer (PB) used in the article refers to the mixture of 19% NaH2 PO4 and 81% Na2 HPO4 unless otherwise specified. The VS-grafted polymers (DX40k-VS, CMDX40k-VS, HA29k-VS and 8arm PEG20k-VS) were dissolved in 0.4 M PB/D2 O, respectively. The stoichiometric ratio of the VS/SH was intentionally controlled to exceed 1.5 such that SH groups could be fully crosslinked with VS groups, and all hydrogel formulations could have a similar crosslinking density. The two components were mixed at a 1:1 volume ratio, and transferred to the bottom of NMR tubes (0.5 ml/tube). After gelation for 18 h at ambient temperature, the well-formed hydrogel samples were covered with a 1.5 ml 0.2 M PB/D2 O buffer to keep the hydrogel fully hydrated, and also to maintain the pH over the whole degradation period. For the measurement of polymer solutions, DX40k-O-SH and unmodified polymers (DX40k, DX20 0 0k and HA29k) were dissolved in 0.2 M PB/D2 O. The 1% DX40k-O-SH consisted DX40k-O-SH only. The 5%, 8% and 12% groups contained mixed DX40k-O-SH and unmodified DX40k at a 1:1 ratio to mimic the composition in the parallel hydrogel formulations at different polymer concentrations. The
5% DX40k-O-SH/native DX20 0 0k and DX40k-O-SH/native HA29k polymer solution mixtures were prepared in the same way. Each hydrogel, or polymer solution formulation, was prepared in triplicate and incubated at 37 °C. The standard 1D 1 H NMR spectra were recorded periodically on a Varian mercury 300 MHz high resolution NMR spectrometer, with a probe temperature at 20 o C. The data was acquired using VNMRJ 2.2D (Agilent, US). Spectral width was 2998.5 Hz, acquisition time was 3.33 s, relaxation delay was set to 1 s unless specified otherwise. Pulse width was 45°. Spectra were processed using ACD/NMR processor 12.01 academic edition (Advanced Chemistry Development, Canada). Chemical shifts are presented in ppm. The ester-neighboring methyl group protons could be clearly distinguished before and after hydrolysis (see Fig. 1). The chemical structure of the esters, as well as the characteristic methyl group associated proton shifts before/after hydrolysis in different formulations is summarized in Table 1. All spectra were calibrated by referencing the characteristic HDO signal peak to 4.75 ppm. The peak area of the ester neighboring methyl protons was integrated for intact/hydrolyzed esters. The total amount of ester (E0 ) was defined as the sum of intact (Et ) and hydrolyzed ester (Eh ) in a specific sample at that time point. 2.5. Acquisition of 1 H NMR spectra in H2 O solvent Hydrolytic kinetics of 1% DX40k-O-SH solutions, and 5% dextran hydrogels were conducted in H2 O solvent using a similar method
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Fig. 1. Representative 1 H NMR spectrum showing the chemical shift of ester groups before and after hydrolysis in (upper) soluble polymer mixture of 5% w/v DX40k/DX40kO-SH; (lower) hydrogel formed by crosslinking 5% w/v DX40k-VS with DX40k-O-SH. The similar spectra of hydrogels formulated using PEG, HA and CM-dextran are provided in the supplementary information.
Table 1 Chemical structure of dextran derivatives via ester linkage, and the chemical shift of ester neighboring CH3 in 1 H NMR spectra using 0.2 M PB/D2 O as solvent.
as previously described for D2 O, with following modifications. The H2 O buffers were prepared with a solvent mixture consisting 10% D2 O/90% H2 O. The 1D 1 H NMR experiments were conducted with presat function to suppress the H2 O signals. Auto-lock and prescan shimming functions were disabled. The intact/hydrolyzed methyl peaks were identified and integrated based on the relative positions and ranges in the corresponding D2 O solvent produced spectra.
2.6. Hydrogel degradation in D2 O and H2 O phosphate buffers The ester-containing DX40k-O-SH was dissolved in D2 O– 20% w/v, and the VS-grafted polymers (DX40k-VS, CMDX40k-VS, HA29k-VS and 8arm PEG20k-VS) were dissolved in 0.4 M PB/D2 O– 20% w/v. The VS- and SH-polymers were mixed at a 1:1 volume ratio. The solution mixture was pipetted on a hydrophobic paraffin film forming hemispherical droplets, each about 40 μL, then
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incubated for 18 h in a humidified chamber at ambient temperature to complete gelation. The weight of the gel was recorded as the initial gel weight (W0 ). The hydrogels were incubated in 1 mL D2 O or H2 O based 0.2 M PB at 37 °C, and were weighed periodically (Mt ). The swelling ratio (Wt /W0 ) was plotted over time until the total dissolution of the gel in the buffer.
Table 2 Hydrolysis rate constant (kobs ) and half-life (t0.5 ) of DX40k-VS/DX40k-O-SH crosslinked hydrogels, as well as the polymer solutions mixed using DX40k-O-SH with other polymers at different polymer concentrations in 0.2 M PB/D2 O at 37 °C. Composition Hydrogel
2.7. Statistical analysis Data were presented as mean ± SD. Ordinary one-way ANOVA test was performed for data analysis (GraphPad Prism 8; GraphPad Software, La Jolla, CA). A statistically significant difference was stated when P < 0.05. 3. Results and discussion In this study, we directly measured the hydrolysis rate constant (kobs ) of hydrolytically degradable hydrogels from the timedependent change of esters obtained from the 1 H NMR spectra. To the best of our knowledge, this is the first demonstration of direct measurement of kobs in hydrogels. The gels were formulated by crosslinking ester-containing DX40k-O-SH with various VSfunctionalized polymers. The 1 H NMR spectroscopy-based method is a useful tool to examine the hydrolysis rate of ester linkers. Importantly, the measurement is not derived from the hydrogel degradation profiles, such as the polymer mass loss profile and the gel swelling profile. According to our published work by stochastic modeling, hydrogel degradation profiles could be altered by manipulating the hydrogel composition, such as polymer concentration and crosslinker functionality, while keeping the kobs values of the ester linkers the same [21]. With the kobs directly and experimentally measured from the degrading hydrogel, the influence of hydrogel composition, as well as the micro-milieu can be evaluated independently. Methacrylate derived esters were chosen in this study due to their clear signals in the 1 H NMR spectra. The characteristic chemical shift of the ester neighboring methyl group can be easily separated from the other peaks, making the quantitative analysis simpler (see Table 1). Van Dijk Wolthuis et al. measured the kobs of dextran-O-MA in its solution form [25]. The hydrogel precursor in this study, DX40kO-SH was synthesized from DX40k-MA, and the ester groups shared similar chemical structures. The hydrolysis kinetics of the esters in DX40k-O-SH solutions, and DX40k-O-SH crosslinked hydrogels was assumed to follow the equation below:
d[Ester] = −kobs [Ester] dt Hence, the value of kobs was calculated from the slope of the initial linear range of −ln EEt versus time (see Fig. 2), where Et and 0 E0 were the amount of intact esters at time t and the total amount of esters, respectively. The Et /E0 ratio was not affected by the relaxation delay (see Table S7 in supplementary information). The halflife of hydrolysis (t0.5 ) was calculated using the equation t0.5 = kln2 . obs
5
[Polymer] kobs (× 10−3 day−1 ) w/v%
DX40k-O-SH crosslinked with DX40k-VS Polymer solution DX40k-O-SH only DX40k-O-SH mixed with DX40k
5 8 12 1 5 8 12 DX40k-O-SH mixed 5 with DX2000k DX40k-O-SH mixed 5 with HA29k
t0.5 (day)
122.1 ± 4.4 131.9 ± 2.6 122.3 ± 3.1 68.8 ± 3.4 68.3 ± 0.9 69.0 ± 0.9 71.7 ± 0.4 64.9 ± 0.4
5.7 ± 0.2 5.3 ± 0.1 5.7 ± 0.1 10.1 ± 0.5 10.1 ± 0.1 10.1 ± 0.1 9.8 ± 0.1 10.7 ± 0.1
65.0 ± 0.6
10.7 ± 0.1
polymers. However, the degree of hydration is expected to be homogeneous for hydrogels formed by hydrophilic polymers, such as dextran, HA or PEG, thus the polymer concentration is expected to have minimal effect on kobs . Our results also showed the value of kobs in hydrogels changed at different stages of hydrolytic degradation. We observed that kobs in hydrogels decreased as degradation proceeded, as evidenced by the decreasing slope in the plots of −ln EEt versus time (see Fig. 2B). For soluble polymers, 0 this slope remained constant, meaning that kobs was the same over the course of degradation. We attribute the decelerated hydrolysis of esters in the degradable hydrogels to the accumulation of carboxylates in the polymer network. A localized, negatively charged environment was built surrounding the intact esters, although the pH of whole system remained constant. The effect of local negative charge on kobs was further studied using HA-VS and CM-dextran-VS, and this will be discussed in a separate section. Ester hydrolysis kinetics were compared between crosslinked polymers (gel state) and soluble polymers (solution state). The value of kobs was sensitive to whether polymers were chemically crosslinked (see Table 2). The values of kobs in gels were roughly 1.8-fold greater in gels than in polymer solutions, and the trend was independent of polymer concentration. Considering that the SH group is 8 atoms away from the ester carbonyl, we argue that the difference should come from the network milieu instead of the Michael addition between SH and VS. Unlike chemical crosslinking, the physical entanglement between polymers had a negligible effect on kobs . The measured kobs in soluble DX40k-O-SH at dilute concentration (1 w/v%) and semi-dilute concentrations (5–12 w/v%), in which polymers chains were entangled, shared similar values (p > 0.05). This observation is consistent with the data reported by van Dijk-Wolthuis et al., which showed that polymer concentration had a minimum influence on kobs [25]. Therefore, we reason that the acceleration of ester hydrolysis in the crosslinked polymers was due to the local network milieu, although the mechanism is unclear. The direct measurement of kobs reveals that the ester hydrolysis rate in soluble polymers does not equal that in crosslinked polymers.
3.1. Influence of polymer concentration and sol/gel state differences on ester hydrolysis kinetics
3.2. Influence of network charge on kobs of DX40k-O-SH hydrogels
Our results showed polymer concentration did not influence the kobs of esters in the hydrogels within the range we studied (see Table 2). The kobs values of DX40k-VS/DX40k-O-SH crosslinked hydrogels at 5%, 8% and 12% w/v were not statistically different (p > 0.05). Polymer concentration has been reported to be a parameter affecting kobs in certain hydrogels with hydrophobic domains, such as the PLA-b-PEG-b-PLA hydrogels [26]. The local water molecules surrounding the esters, which are adjacent to hydrophobic PLA domains, are sensitive to the concentration of
Compared to hydrogels formulated with neutral polymers (DXVS and PEG-VS), ester hydrolysis proceeded slower in hydrogels formed with negatively charged HA-VS and CMDX-VS polymers (see Table 3). DX40k-O-SH was crosslinked with four types of VSfunctionalized polymer, namely, (1) DX40k-VS; (2) 8-arm PEG20kVS; (3) HA29k-VS and (4) CMDX40k-VS. The former two were neutral in charge and the latter two carried negatively charged carboxylates in the polymer backbones. Since the hydrolysis of DXO-MA species is mainly catalyzed by OH(D)− at physiological pH
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Fig. 2. Representative plots showing the hydrolysis kinetics of esters in 0.2 M PB/D2 O at 37 °C. (A): DX40k-O-SH solutions at different concentrations; (B):Hydrogels formed by crosslinking DX40k-VS and DX40k-O-SH at different concentrations; (C): Hydrogels varied in initial network charge, formed by crosslinking DX40k-O-SH with DX40k-VS (neutral); 8-arm PEG20k-VS (neutral); HA20k-VS (negative); CMDX40k-VS (negative). Table 3 Hydrolysis rate constant (kobs ) and half-life of hydrogel formulations using DX40k-O-SH to crosslink different–VS functionalized polymers in 0.2 M PB/D2 O at 37 °C. Formulation
[Polymer] w/v%
kobs (× 10−3 day−1 )
t0.5 (day)
Polymer charge
1 2 3 4
5
124.5 ± 2.8 122.1 ± 4.4 78.6 ± 4.7 81.3 ± 1.6
5.6 ± 0.1 5.7 ± 0.2 8.8 ± 0.5 8.5 ± 0.2
Neutral
8-arm PEG20k-VS crosslinked with DX40k-O-SH DX40k-VS crosslinked with DX40k-O-SH HA29k-VS crosslinked with DX40k-O-SH CMDX40k-VS crosslinked with DX40k-O-SH
(∼7.4) [25], we hypothesize that the carboxylates insulated the esters in close proximity from the OH(D)− attack. As hydrogel degradation proceeded, the accumulation of hydrolysis product carboxylates would exhibit an increasing effect inhibiting the neighboring esters from hydrolysis. Such a mechanism explains the changing kobs in different stages of hydrogel degradation. The charge effect of neighboring groups has been reported and utilized in the tuning ester hydrolysis rate of polymer-drug conjugates [32] and hydrogels [23]. Jo et al. used peptide linkers with positively charged arginine and negatively charged aspartate to, respectively, accelerate and inhibit the hydrolysis of neighboring esters [23]. In our experiments, we observed that the remote charged groups on a polymer network cast a similar neighboring group participation effect, and the effect was limited only to the gel state. The kobs values of esters in a polymer solution mixture of HA29k and DX40k-O-SH did not appear to be influenced by charge (see Table 2). In addition to the direct measurement of kobs using 1 H NMR spectroscopy, which focuses on the cleavage of each ester bond in the polymer network, we also followed the swelling profiles of hydrogels in 0.2 M PB buffers as an indirect indication of polymer network disintegration in bulk (see Fig. 3A). The polymer concentrations for all formulations were increased to 20% w/v to strengthen the gels for easier handling. A similar swelling ratio at
Negative
the equilibrium state of hydrogels formulated using DX40k-VS (see Fig. 3A–2), HA29k (see Fig. 3A–3) and CMDX40k (see Fig. 3A–4) suggested these gels had a similar initial crosslinking density. The overall trend of gel dissolution time echoed the kobs data: the hydrogels composed of negatively charged polymers degraded slower than those of neutral polymers. However, the PEG-consisting gels (see Fig. 3A–1) collapsed earlier than the dextran gels (see Fig. 3A– 2), although the kobs values of these two formulations were the same (see Table 3). The 8-arm PEG20k-VS is a highly branched, star-shaped polymer, and the VS groups were located at the end of each arm, while the VS groups were grafted along the linear backbone of other polymers. The PEG gels had a higher initial equilibrium swelling ratio than the other formulations, indicating that the crosslinking densities in the PEG gels were lower than the others. Therefore, the PEG gels apparently collapsed faster than the dextran gels, although they had the same rate of hydrolytic degradation. These examples well demonstrated the influence of the local milieu (such as charge) and polymer structure (such as shape) on the apparent “hydrogel degradation profile”. With the direct measurement of kobs in the hydrogels, we could decouple the hydrolytic cleavage rate of ester linkages from other factors, so that the real influence of these factors was revealed and the existing models can then be improved.
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Fig. 3. Swelling profiles of hydrolysable hydrogels (20% w/v) at 37 °C in 0.2 M PB using (A) D2 O and (B) H2 O as solvent. All hydrogels were formed by crosslinking DX40k-OSH to VS functionalized polymers via Michael addition. Formulation: (1) DX40k-O-SH + 8-arm PEG20k-VS; (2) DX40k-O-SH + DX40k-VS; (3) DX40k-O-SH + HA29k-VS; (4) DX40k-O-SH + CMDX40k-VS.
Fig. 4. Comparison of deuterium solvent effect on hydrolysis kinetics of esters in (Left): 1% w/v DX40k-O-SH solution; (Right) hydrogels formed by crosslinking DX40k-VS and DX40k-O-SH at 5% w/v. Phosphate buffer concentration was 0.2 M, samples were incubated at 37 °C.
3.3. Influence of deuterium oxide on kobs It should be noted that the kobs values measured in D2 O based buffers do not reflect the exact value in H2 O environments. Deuterium effect has been well documented to have significant impact on ester hydrolysis [33–35]. although the absolute values of kobs are expected to be different, we hypothese that the hydrolytic kinetics in the D2 O based experiments reflected the same trend as in the H2 O environment. A simple, qualitative assay was conducted to demonstrate the difference. The swelling profiles of all hydrogel formulations were followed in parallel in D2 O and H2 O based 0.2 M PB buffers (see Fig. 3B). All hydrogel formulations degradation profiles in 0.2 M PB/H2 O followed the same trend as in D2 O,
in spite of a faster rate. Further, kobs in the H2 O based buffers (in a mixture 10% D2 O plus 90% H2 O as solvent) were measured directly using modified 1 H NMR. The results indicated a 1.8-fold rate increase for polymer solution, and a 2-fold rate increase for the hydrogel (see Table 4 and Fig. 4). The ratio is consistent with the literature reported trends. Bruice et al. and Lundberg et al. reported that base-catalyzed ester hydrolysis proceeded 2–5 times slower in D2 O-based buffers due to the isotope effect [33,36]. Another factor is that the actual [OD− ] is lower than the [OH− ] in the corresponding D2 O/H2 O buffer at the same temperature. Given the pKw of D2 O and H2 O at 25 °C are 15.0 and 14.0, respectively [37,38], the concentration ratio of OD− /OH− was about 0.36 at 25 °C in the 0.4 M PB buffers being used in this study.
Table 4 Comparison of deuterium solvent effect on hydrolysis rate constant (kobs ) of DX40k-O-SH in the form of solution and hydrogel. Samples were incubated at 37 °C in 0.2 M PB in D2O and 10% D2 O/90% H2 O, respectively.
Solution Hydrogel
Composition
Polymer conc. %w/v
kobs in D2 O n(× 10−3 day−1 )
kobs in H2 O (× 10−3 day−1 )
kobs (H2 O ) kobs (D2 O )
DX40k-O-SH mixed with DX40k DX40k-O-SH crosslinked with DX40k-VS
5 5
68.3 ± 0.9 122.1 ± 4.4
124.1 ± 2.4 248.2 ± 4.7
1.8 2.0
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4. Conclusion This work reports a simple, direct measurement of the hydrolysis of esters in degradable hydrogels using 1 H NMR spectroscopy. The method allows decoupling of the rate of ester cleavage (kobs ) from other factors that can contribute to macroscopic differences in hydrogel degradation. The value of kobs in the hydrogel was found to be sensitive to the milieu of the polymer network in the following respects: (1) The esters were hydrolyzed faster in a chemically crosslinked polymer network than in soluble polymers at equivalent conditions; (2) Ester hydrolysis kinetics were dynamic during the gel-sol phase transition, the kobs decreased with the extent of hydrogel degradation; (3) a negatively charged polymer network inhibited the esters from being hydrolyzed in the polymer network in the hydrogel; and (4) solvent had a significant effect on ester hydrolysis kinetics, with the equivalent kobs in an H2 O environment about 1.8–2-fold greater than the NMRmeasured values in D2 O. Acknowledgment The authors gratefully acknowledge the Hong Kong General Research Fund (GRF16322016) and Science and Technology Plan of Shenzhen (JCY20170818114038319) for supporting the research work. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.actbio.2019.10.030. References [1] A. Vashist, Y.K. Gupta, S. Ahmad, Recent advances in hydrogel based drug delivery systems for the human body, J. Mater. Chem. B 2 (2) (2014) 147–166. [2] E. Caló, V.V. Khutoryanskiy, Biomedical applications of hydrogels: a review of patents and commercial products, Eur. Polym. J. 65 (2015) 252–267. [3] T. Vermonden, R. Censi, W.E. Hennink, Hydrogels for protein delivery, Chem. Rev. 112 (5) (2012) 2853–2888. [4] A.S. Hoffman, Hydrogels for biomedical applications, Adv. Drug Deliv. Rev. 54 (1) (2002) 3–12. [5] S.P. Zustiak, J.B. Leach, Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties, Biomacromolecules 11 (5) (2010) 1348–1357. [6] G.T. Chao, L.Y. Fan, Y. Lin, W.J. Jia, Z.Y. Qian, Y.C. Gu, C.B. Liu, X.P. Ni, J. Li, H.X. Deng, C.Y. Gong, M.L. Gou, K. Lei, A.L. Huang, C.H. Huang, J.L. Yang, B. Kan, M.J. Tu, Synthesis, characterization and hydrolytic degradation of degradable poly(butylene terephthalate)/poly(ethylene glycol) (PBT/PEG) copolymers, J. Mater. Sci. Mater. Med. 18 (3) (2007) 449–455. [7] C. Engineer, J. Parikh, A. Raval, Review on hydrolytic degradation behavior of biodegradable polymers from controlled drug delivery system, Trends Biomater. Artif. Organs 25 (2011) 79–85. [8] C. Hiemstra, L.J. Van Der Aa, Z. Zhong, P.J. Dijkstra, J. Feijen, Novel in situ forming, degradable dextran hydrogels by michael addition chemistry: synthesis, rheology, and degradation, Macromolecules 40 (2007) 1165–1173. [9] W.Van Dijk-Wolthuis, W.N.E. E.VanDijk-Wolthuis, J.A.M. M.Hoogeboom, M.J.V. anSteenbergen, S.K.Y.Y. Tsang, W.E. Hennink, Degradation and release behavior of dextran-based hydrogels, Macromolecules 9297 (97) (1997) 4639–4645. [10] W.N.E. Van Dijk-Wolthuis, O. Franssen, H. Talsma, M.J. Van Steenbergen, J.J. Kettenes-van den Bosch, W.E. Hennink, Synthesis, characterization, and polymerization of glycidyl methacrylate derivatized dextran, Macromolecules 28 (18) (1995) 6317–6322. [11] J.L. West, J.A. Hubbell, Polymeric biomaterials with degradation sites for proteases involved in cell migration, Macromolecules 32 (1) (1999) 241–244. [12] J.A. Burdick, C. Chung, X. Jia, M.A. Randolph, R. Langer, Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks, Biomacromolecules 6 (1) (2005) 386–391.
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Local environment-dependent kinetics of ester hydrolysis revealed by direct 1 H NMR measurement of degrading hydrogels Chi Ming Laurence Lau a,b, Ghodsiehsadat Jahanmir a,b, Ying Chau a,b,∗ a b
The Hong Kong University of Science and Technology Shenzhen Research Institute, Shenzhen, China Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong, China
a r t i c l e
i n f o
Article history: Received 31 May 2019 Revised 5 October 2019 Accepted 23 October 2019 Available online xxx Keywords: Hydrogel Ester hydrolysis Degradable NMR Dextran Peg Hyaluronic acid
a b s t r a c t We have demonstrated the use of a simple 1 H NMR spectrometry-based method to directly measure the pseudo first-order hydrolytic cleavage rate constant (kobs ) of methacrylate-derived ester crosslinkers in hydrogels composed of PEG, dextran, carboxymethyl dextran (CM-dextran) and hyaluronic acid (HA). Using this technique, we systematically examined how the local environment in the hydrogel influenced the rate of ester hydrolysis. Within the formulations being studied, the esters in the crosslinked polymer network (gel state) degraded 1.8 times faster than esters of similar chemistry in soluble polymers (solution state). Furthermore, the value of kobs was independent of the polymer concentration or the hydrogel network structure, although these parameters affected the swelling profiles in response to the hydrolytic degradation. On the other hand, the presence of the negatively charged carboxylate groups in the polymer chains decreased kobs in gel state, while only minimally affecting kobs in solution state. Hydrogels composed of negatively charged polymers (HA and CM-dextran) had a kobs about 30% smaller than hydrogels composed of neutral polymers (dextran and PEG). The reported method provides a reliable tool to resolve conflicting views about hydrogel degradation, and to guide the rational design of degradable hydrogel. Statement of significance Degradable hydrogels are widely used in biological applications. A common degradation mechanism of the crosslinked polymer is by hydrolytic cleavage. However, the hydrogel micro-milieu do affect the behavior of the hydrolysable bonds, for example esters. There have been several conflicting speculations on how hydrogel composition would affect the macroscopic degradation behavior. In this report, we simply, but innovatively applied ordinary 1 H NMR spectrometry-based method to probe the rate of ester cleavage in the native hydrogel milieu. We tried to answer whether these parameters will have direct influence on ester cleavage, or have indirect effect on the overall network disintegration behavior. This study provides quantitative evidences to assist theoretical modeling and to guide rational formulation design. © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction A hydrogel is a water-retaining network composed of hydrophilic polymers in an aqueous environment. The unique properties of a hydrogel have attracted intensive research about its potential applications in biomedical areas [1–4]. For many applications, a predictable and controllable degradation profile is advantageous. Formation of the hydrogel network is via chemical or physical crosslinking between precursor polymers, and/or ∗ Corresponding author at: The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China. E-mail address: [email protected] (Y. Chau).
monomers, and hydrogel degradation can be achieved by hydrolysis [5–10], enzymatic digestion [11,12], or photolysis [13,14] of the polymer network. Commonly utilized hydrolysable moieties include anhydrides, esters and amides, among which esters have a moderate hydrolytic half-life (at the scale of days) under physiological conditions. Since the polymer network in hydrogel is highly hydrated, bulk degradation dominates the erosion of most hydrolytically degradable hydrogels [15]. Thorough understanding of the influence of hydrogel parameters on the macroscopic degradation profile would be extremely helpful to guide the rational formulation design. However, these parameters are usually interdependent, making the process of bulk degradation too complex to be described using simple equations. Theoretical modelling
https://doi.org/10.1016/j.actbio.2019.10.030 1742-7061/© 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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approaches, such as the mass loss model [16,17] and equilibrium swelling-model [9] are widely used to describe and predict the macroscopic degradation phenomena of hydrolysable hydrogels quantitatively [18–23]. In general, the models adopt a two-step approach: construct a numeric polymer network first, then simulate the process of network disintegration and calculate the corresponding output parameters. In the first step, the initial properties of the polymer network, such as the crosslinking density and distribution of degradable bonds in the network, are calculated based on the linker functionality (number of crosslinkers per chain) and polymer concentration (the number of polymer chains). Polymer crosslinking is often assumed ideal, although certain more sophisticated models also include non-idealities in the algorithm [18–21]. In the second step, a rate of network dissociation is assigned to the cleavable ester linkages in hydrolysable hydrogels, and models will generate measurable parameters such as polymer mass loss, and/or gel swelling ratios as outputs. The hydrolytic cleavage kinetics is described using a pseudo first-order rate constant (kobs ). The kobs is a lumped parameter that includes the external environmental parameters, such as temperature, pH, ionic strength etc. The values of ester kobs are usually indirectly measured by HPLC or NMR based methods in soluble hydrogel precursors, and generalized to gel state at which the precursors are crosslinked. In limited cases, kobs in hydrogel can be directly determined by following the release profile of ester-conjugated moieties from the hydrogel [23]. However, assuming an equal kobs value before and after gelation lacks experimental evidences. There have been some conflicting speculations about whether the kobs of an ester is the same in hydrogel formulations that differ in degree of network crosslinking [24], polymer concentration [21,25], and the network charge [23]. Several studies have reported that hydrogel degradation behavior varied significantly in different formulations, although the esters shared the same chemistry [9,16,25,26]. The observations indicate that kobs may be hydrogel milieu-dependent. However, it is also possible that the swelling profile and disintegration time can be significantly changed by altering polymer concentration only, with the kobs remaining unchanged [21]. On the other hand, the value of kobs is often assumed to be constant through the degradation process if the external environmental parameters do not change, which may not be true. Since the hydrogel structural parameters are changing in a degrading hydrogel, we anticipate that kobs will change during hydrogel degradation. Therefore, a technique to determine the kobs in the specific hydrogel environment is valuable to resolve the conflicting views about hydrogel degradation. Here, we adopt a simple method using conventional 1 H NMR spectroscopy to directly measure the kobs of the ester crosslinkers in hydrogels. The 1 H NMR has been routinely used in hydrolysis rate measurement for different materials in solution state [16,27]. For certain ester species, the signature shift of carbonyl neighboring protons can be easily differentiated before and after hydrolysis. The hydrolysis kinetics can be followed by tracking the rate of consumption of intact esters, or the generation of hydrolyzed esters. In this study, we have extended the method to the gel state, in which the polymers are covalently crosslinked. To demonstrate the utility of this approach, hydrolytically degradable hydrogels were formulated using various common polymers, including polyethylene glycol (PEG), dextran and hyaluronic acid (HA). The values of kobs were determined for formulations varying in polymer concentration, polymer conformation and crosslinker functionality, as well as the network charge. Since the method allows direct measurement of kobs in gel state, the contribution of the hydrogel’s local environment can be systematically studied.
2. Material and methods 2.1. Synthesis of vinyl sulfone functionalized dextran (DX40k-VS), carboxymethyl-dextran (CMDX40k-VS) and hyaluronic acid (HA29k-VS) The 8-arm PEG20k-VS (95%) was purchased from JenKem Technology, USA. Carboxymethyl-dextran-40k (CMDX-40k) was prepared based on a previously reported method [28]. Vinyl sulfone (VS)-functionalized hydroxyl-rich polysaccharides were synthesized as previously reported [29]. In brief, polymers were dissolved in dilute sodium hydroxide solution at desired concentrations. Divinyl sulfone (97% contains < 650 ppm hydroquinone as inhibitor, Aldrich) was added in excess (Scheme 1). The degree of VS conjugation was controlled by reaction time, and the reaction was stopped by pH decrease (
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3
Scheme 1. Conjugation of vinyl sulfone (VS) to hydroxyl rich polysaccharides.
Scheme 2. Conjugation of thiol group to dextran via ester linkers.
NaH2 PO4 and Na2 HPO4 stock solutions at different ratios (details are provided in the Table S5 in supplementary information). The apparent pH values were measured using an Orion micro pH electrode with Star A111 benchtop pH meter (Thermo-scientific). The value of pD can be quickly approximated by adding an empirical conversion equation [31]:
pD = pHa + 0.4 The phosphate buffer (PB) used in the article refers to the mixture of 19% NaH2 PO4 and 81% Na2 HPO4 unless otherwise specified. The VS-grafted polymers (DX40k-VS, CMDX40k-VS, HA29k-VS and 8arm PEG20k-VS) were dissolved in 0.4 M PB/D2 O, respectively. The stoichiometric ratio of the VS/SH was intentionally controlled to exceed 1.5 such that SH groups could be fully crosslinked with VS groups, and all hydrogel formulations could have a similar crosslinking density. The two components were mixed at a 1:1 volume ratio, and transferred to the bottom of NMR tubes (0.5 ml/tube). After gelation for 18 h at ambient temperature, the well-formed hydrogel samples were covered with a 1.5 ml 0.2 M PB/D2 O buffer to keep the hydrogel fully hydrated, and also to maintain the pH over the whole degradation period. For the measurement of polymer solutions, DX40k-O-SH and unmodified polymers (DX40k, DX20 0 0k and HA29k) were dissolved in 0.2 M PB/D2 O. The 1% DX40k-O-SH consisted DX40k-O-SH only. The 5%, 8% and 12% groups contained mixed DX40k-O-SH and unmodified DX40k at a 1:1 ratio to mimic the composition in the parallel hydrogel formulations at different polymer concentrations. The
5% DX40k-O-SH/native DX20 0 0k and DX40k-O-SH/native HA29k polymer solution mixtures were prepared in the same way. Each hydrogel, or polymer solution formulation, was prepared in triplicate and incubated at 37 °C. The standard 1D 1 H NMR spectra were recorded periodically on a Varian mercury 300 MHz high resolution NMR spectrometer, with a probe temperature at 20 o C. The data was acquired using VNMRJ 2.2D (Agilent, US). Spectral width was 2998.5 Hz, acquisition time was 3.33 s, relaxation delay was set to 1 s unless specified otherwise. Pulse width was 45°. Spectra were processed using ACD/NMR processor 12.01 academic edition (Advanced Chemistry Development, Canada). Chemical shifts are presented in ppm. The ester-neighboring methyl group protons could be clearly distinguished before and after hydrolysis (see Fig. 1). The chemical structure of the esters, as well as the characteristic methyl group associated proton shifts before/after hydrolysis in different formulations is summarized in Table 1. All spectra were calibrated by referencing the characteristic HDO signal peak to 4.75 ppm. The peak area of the ester neighboring methyl protons was integrated for intact/hydrolyzed esters. The total amount of ester (E0 ) was defined as the sum of intact (Et ) and hydrolyzed ester (Eh ) in a specific sample at that time point. 2.5. Acquisition of 1 H NMR spectra in H2 O solvent Hydrolytic kinetics of 1% DX40k-O-SH solutions, and 5% dextran hydrogels were conducted in H2 O solvent using a similar method
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Fig. 1. Representative 1 H NMR spectrum showing the chemical shift of ester groups before and after hydrolysis in (upper) soluble polymer mixture of 5% w/v DX40k/DX40kO-SH; (lower) hydrogel formed by crosslinking 5% w/v DX40k-VS with DX40k-O-SH. The similar spectra of hydrogels formulated using PEG, HA and CM-dextran are provided in the supplementary information.
Table 1 Chemical structure of dextran derivatives via ester linkage, and the chemical shift of ester neighboring CH3 in 1 H NMR spectra using 0.2 M PB/D2 O as solvent.
as previously described for D2 O, with following modifications. The H2 O buffers were prepared with a solvent mixture consisting 10% D2 O/90% H2 O. The 1D 1 H NMR experiments were conducted with presat function to suppress the H2 O signals. Auto-lock and prescan shimming functions were disabled. The intact/hydrolyzed methyl peaks were identified and integrated based on the relative positions and ranges in the corresponding D2 O solvent produced spectra.
2.6. Hydrogel degradation in D2 O and H2 O phosphate buffers The ester-containing DX40k-O-SH was dissolved in D2 O– 20% w/v, and the VS-grafted polymers (DX40k-VS, CMDX40k-VS, HA29k-VS and 8arm PEG20k-VS) were dissolved in 0.4 M PB/D2 O– 20% w/v. The VS- and SH-polymers were mixed at a 1:1 volume ratio. The solution mixture was pipetted on a hydrophobic paraffin film forming hemispherical droplets, each about 40 μL, then
Please cite this article as: C.M.L. Lau, G. Jahanmir and Y. Chau, Local environment-dependent kinetics of ester hydrolysis revealed by direct 1 H NMR measurement of degrading hydrogels, Acta Biomaterialia, https://doi.org/10.1016/j.actbio.2019.10.030
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incubated for 18 h in a humidified chamber at ambient temperature to complete gelation. The weight of the gel was recorded as the initial gel weight (W0 ). The hydrogels were incubated in 1 mL D2 O or H2 O based 0.2 M PB at 37 °C, and were weighed periodically (Mt ). The swelling ratio (Wt /W0 ) was plotted over time until the total dissolution of the gel in the buffer.
Table 2 Hydrolysis rate constant (kobs ) and half-life (t0.5 ) of DX40k-VS/DX40k-O-SH crosslinked hydrogels, as well as the polymer solutions mixed using DX40k-O-SH with other polymers at different polymer concentrations in 0.2 M PB/D2 O at 37 °C. Composition Hydrogel
2.7. Statistical analysis Data were presented as mean ± SD. Ordinary one-way ANOVA test was performed for data analysis (GraphPad Prism 8; GraphPad Software, La Jolla, CA). A statistically significant difference was stated when P < 0.05. 3. Results and discussion In this study, we directly measured the hydrolysis rate constant (kobs ) of hydrolytically degradable hydrogels from the timedependent change of esters obtained from the 1 H NMR spectra. To the best of our knowledge, this is the first demonstration of direct measurement of kobs in hydrogels. The gels were formulated by crosslinking ester-containing DX40k-O-SH with various VSfunctionalized polymers. The 1 H NMR spectroscopy-based method is a useful tool to examine the hydrolysis rate of ester linkers. Importantly, the measurement is not derived from the hydrogel degradation profiles, such as the polymer mass loss profile and the gel swelling profile. According to our published work by stochastic modeling, hydrogel degradation profiles could be altered by manipulating the hydrogel composition, such as polymer concentration and crosslinker functionality, while keeping the kobs values of the ester linkers the same [21]. With the kobs directly and experimentally measured from the degrading hydrogel, the influence of hydrogel composition, as well as the micro-milieu can be evaluated independently. Methacrylate derived esters were chosen in this study due to their clear signals in the 1 H NMR spectra. The characteristic chemical shift of the ester neighboring methyl group can be easily separated from the other peaks, making the quantitative analysis simpler (see Table 1). Van Dijk Wolthuis et al. measured the kobs of dextran-O-MA in its solution form [25]. The hydrogel precursor in this study, DX40kO-SH was synthesized from DX40k-MA, and the ester groups shared similar chemical structures. The hydrolysis kinetics of the esters in DX40k-O-SH solutions, and DX40k-O-SH crosslinked hydrogels was assumed to follow the equation below:
d[Ester] = −kobs [Ester] dt Hence, the value of kobs was calculated from the slope of the initial linear range of −ln EEt versus time (see Fig. 2), where Et and 0 E0 were the amount of intact esters at time t and the total amount of esters, respectively. The Et /E0 ratio was not affected by the relaxation delay (see Table S7 in supplementary information). The halflife of hydrolysis (t0.5 ) was calculated using the equation t0.5 = kln2 . obs
5
[Polymer] kobs (× 10−3 day−1 ) w/v%
DX40k-O-SH crosslinked with DX40k-VS Polymer solution DX40k-O-SH only DX40k-O-SH mixed with DX40k
5 8 12 1 5 8 12 DX40k-O-SH mixed 5 with DX2000k DX40k-O-SH mixed 5 with HA29k
t0.5 (day)
122.1 ± 4.4 131.9 ± 2.6 122.3 ± 3.1 68.8 ± 3.4 68.3 ± 0.9 69.0 ± 0.9 71.7 ± 0.4 64.9 ± 0.4
5.7 ± 0.2 5.3 ± 0.1 5.7 ± 0.1 10.1 ± 0.5 10.1 ± 0.1 10.1 ± 0.1 9.8 ± 0.1 10.7 ± 0.1
65.0 ± 0.6
10.7 ± 0.1
polymers. However, the degree of hydration is expected to be homogeneous for hydrogels formed by hydrophilic polymers, such as dextran, HA or PEG, thus the polymer concentration is expected to have minimal effect on kobs . Our results also showed the value of kobs in hydrogels changed at different stages of hydrolytic degradation. We observed that kobs in hydrogels decreased as degradation proceeded, as evidenced by the decreasing slope in the plots of −ln EEt versus time (see Fig. 2B). For soluble polymers, 0 this slope remained constant, meaning that kobs was the same over the course of degradation. We attribute the decelerated hydrolysis of esters in the degradable hydrogels to the accumulation of carboxylates in the polymer network. A localized, negatively charged environment was built surrounding the intact esters, although the pH of whole system remained constant. The effect of local negative charge on kobs was further studied using HA-VS and CM-dextran-VS, and this will be discussed in a separate section. Ester hydrolysis kinetics were compared between crosslinked polymers (gel state) and soluble polymers (solution state). The value of kobs was sensitive to whether polymers were chemically crosslinked (see Table 2). The values of kobs in gels were roughly 1.8-fold greater in gels than in polymer solutions, and the trend was independent of polymer concentration. Considering that the SH group is 8 atoms away from the ester carbonyl, we argue that the difference should come from the network milieu instead of the Michael addition between SH and VS. Unlike chemical crosslinking, the physical entanglement between polymers had a negligible effect on kobs . The measured kobs in soluble DX40k-O-SH at dilute concentration (1 w/v%) and semi-dilute concentrations (5–12 w/v%), in which polymers chains were entangled, shared similar values (p > 0.05). This observation is consistent with the data reported by van Dijk-Wolthuis et al., which showed that polymer concentration had a minimum influence on kobs [25]. Therefore, we reason that the acceleration of ester hydrolysis in the crosslinked polymers was due to the local network milieu, although the mechanism is unclear. The direct measurement of kobs reveals that the ester hydrolysis rate in soluble polymers does not equal that in crosslinked polymers.
3.1. Influence of polymer concentration and sol/gel state differences on ester hydrolysis kinetics
3.2. Influence of network charge on kobs of DX40k-O-SH hydrogels
Our results showed polymer concentration did not influence the kobs of esters in the hydrogels within the range we studied (see Table 2). The kobs values of DX40k-VS/DX40k-O-SH crosslinked hydrogels at 5%, 8% and 12% w/v were not statistically different (p > 0.05). Polymer concentration has been reported to be a parameter affecting kobs in certain hydrogels with hydrophobic domains, such as the PLA-b-PEG-b-PLA hydrogels [26]. The local water molecules surrounding the esters, which are adjacent to hydrophobic PLA domains, are sensitive to the concentration of
Compared to hydrogels formulated with neutral polymers (DXVS and PEG-VS), ester hydrolysis proceeded slower in hydrogels formed with negatively charged HA-VS and CMDX-VS polymers (see Table 3). DX40k-O-SH was crosslinked with four types of VSfunctionalized polymer, namely, (1) DX40k-VS; (2) 8-arm PEG20kVS; (3) HA29k-VS and (4) CMDX40k-VS. The former two were neutral in charge and the latter two carried negatively charged carboxylates in the polymer backbones. Since the hydrolysis of DXO-MA species is mainly catalyzed by OH(D)− at physiological pH
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Fig. 2. Representative plots showing the hydrolysis kinetics of esters in 0.2 M PB/D2 O at 37 °C. (A): DX40k-O-SH solutions at different concentrations; (B):Hydrogels formed by crosslinking DX40k-VS and DX40k-O-SH at different concentrations; (C): Hydrogels varied in initial network charge, formed by crosslinking DX40k-O-SH with DX40k-VS (neutral); 8-arm PEG20k-VS (neutral); HA20k-VS (negative); CMDX40k-VS (negative). Table 3 Hydrolysis rate constant (kobs ) and half-life of hydrogel formulations using DX40k-O-SH to crosslink different–VS functionalized polymers in 0.2 M PB/D2 O at 37 °C. Formulation
[Polymer] w/v%
kobs (× 10−3 day−1 )
t0.5 (day)
Polymer charge
1 2 3 4
5
124.5 ± 2.8 122.1 ± 4.4 78.6 ± 4.7 81.3 ± 1.6
5.6 ± 0.1 5.7 ± 0.2 8.8 ± 0.5 8.5 ± 0.2
Neutral
8-arm PEG20k-VS crosslinked with DX40k-O-SH DX40k-VS crosslinked with DX40k-O-SH HA29k-VS crosslinked with DX40k-O-SH CMDX40k-VS crosslinked with DX40k-O-SH
(∼7.4) [25], we hypothesize that the carboxylates insulated the esters in close proximity from the OH(D)− attack. As hydrogel degradation proceeded, the accumulation of hydrolysis product carboxylates would exhibit an increasing effect inhibiting the neighboring esters from hydrolysis. Such a mechanism explains the changing kobs in different stages of hydrogel degradation. The charge effect of neighboring groups has been reported and utilized in the tuning ester hydrolysis rate of polymer-drug conjugates [32] and hydrogels [23]. Jo et al. used peptide linkers with positively charged arginine and negatively charged aspartate to, respectively, accelerate and inhibit the hydrolysis of neighboring esters [23]. In our experiments, we observed that the remote charged groups on a polymer network cast a similar neighboring group participation effect, and the effect was limited only to the gel state. The kobs values of esters in a polymer solution mixture of HA29k and DX40k-O-SH did not appear to be influenced by charge (see Table 2). In addition to the direct measurement of kobs using 1 H NMR spectroscopy, which focuses on the cleavage of each ester bond in the polymer network, we also followed the swelling profiles of hydrogels in 0.2 M PB buffers as an indirect indication of polymer network disintegration in bulk (see Fig. 3A). The polymer concentrations for all formulations were increased to 20% w/v to strengthen the gels for easier handling. A similar swelling ratio at
Negative
the equilibrium state of hydrogels formulated using DX40k-VS (see Fig. 3A–2), HA29k (see Fig. 3A–3) and CMDX40k (see Fig. 3A–4) suggested these gels had a similar initial crosslinking density. The overall trend of gel dissolution time echoed the kobs data: the hydrogels composed of negatively charged polymers degraded slower than those of neutral polymers. However, the PEG-consisting gels (see Fig. 3A–1) collapsed earlier than the dextran gels (see Fig. 3A– 2), although the kobs values of these two formulations were the same (see Table 3). The 8-arm PEG20k-VS is a highly branched, star-shaped polymer, and the VS groups were located at the end of each arm, while the VS groups were grafted along the linear backbone of other polymers. The PEG gels had a higher initial equilibrium swelling ratio than the other formulations, indicating that the crosslinking densities in the PEG gels were lower than the others. Therefore, the PEG gels apparently collapsed faster than the dextran gels, although they had the same rate of hydrolytic degradation. These examples well demonstrated the influence of the local milieu (such as charge) and polymer structure (such as shape) on the apparent “hydrogel degradation profile”. With the direct measurement of kobs in the hydrogels, we could decouple the hydrolytic cleavage rate of ester linkages from other factors, so that the real influence of these factors was revealed and the existing models can then be improved.
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Fig. 3. Swelling profiles of hydrolysable hydrogels (20% w/v) at 37 °C in 0.2 M PB using (A) D2 O and (B) H2 O as solvent. All hydrogels were formed by crosslinking DX40k-OSH to VS functionalized polymers via Michael addition. Formulation: (1) DX40k-O-SH + 8-arm PEG20k-VS; (2) DX40k-O-SH + DX40k-VS; (3) DX40k-O-SH + HA29k-VS; (4) DX40k-O-SH + CMDX40k-VS.
Fig. 4. Comparison of deuterium solvent effect on hydrolysis kinetics of esters in (Left): 1% w/v DX40k-O-SH solution; (Right) hydrogels formed by crosslinking DX40k-VS and DX40k-O-SH at 5% w/v. Phosphate buffer concentration was 0.2 M, samples were incubated at 37 °C.
3.3. Influence of deuterium oxide on kobs It should be noted that the kobs values measured in D2 O based buffers do not reflect the exact value in H2 O environments. Deuterium effect has been well documented to have significant impact on ester hydrolysis [33–35]. although the absolute values of kobs are expected to be different, we hypothese that the hydrolytic kinetics in the D2 O based experiments reflected the same trend as in the H2 O environment. A simple, qualitative assay was conducted to demonstrate the difference. The swelling profiles of all hydrogel formulations were followed in parallel in D2 O and H2 O based 0.2 M PB buffers (see Fig. 3B). All hydrogel formulations degradation profiles in 0.2 M PB/H2 O followed the same trend as in D2 O,
in spite of a faster rate. Further, kobs in the H2 O based buffers (in a mixture 10% D2 O plus 90% H2 O as solvent) were measured directly using modified 1 H NMR. The results indicated a 1.8-fold rate increase for polymer solution, and a 2-fold rate increase for the hydrogel (see Table 4 and Fig. 4). The ratio is consistent with the literature reported trends. Bruice et al. and Lundberg et al. reported that base-catalyzed ester hydrolysis proceeded 2–5 times slower in D2 O-based buffers due to the isotope effect [33,36]. Another factor is that the actual [OD− ] is lower than the [OH− ] in the corresponding D2 O/H2 O buffer at the same temperature. Given the pKw of D2 O and H2 O at 25 °C are 15.0 and 14.0, respectively [37,38], the concentration ratio of OD− /OH− was about 0.36 at 25 °C in the 0.4 M PB buffers being used in this study.
Table 4 Comparison of deuterium solvent effect on hydrolysis rate constant (kobs ) of DX40k-O-SH in the form of solution and hydrogel. Samples were incubated at 37 °C in 0.2 M PB in D2O and 10% D2 O/90% H2 O, respectively.
Solution Hydrogel
Composition
Polymer conc. %w/v
kobs in D2 O n(× 10−3 day−1 )
kobs in H2 O (× 10−3 day−1 )
kobs (H2 O ) kobs (D2 O )
DX40k-O-SH mixed with DX40k DX40k-O-SH crosslinked with DX40k-VS
5 5
68.3 ± 0.9 122.1 ± 4.4
124.1 ± 2.4 248.2 ± 4.7
1.8 2.0
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4. Conclusion This work reports a simple, direct measurement of the hydrolysis of esters in degradable hydrogels using 1 H NMR spectroscopy. The method allows decoupling of the rate of ester cleavage (kobs ) from other factors that can contribute to macroscopic differences in hydrogel degradation. The value of kobs in the hydrogel was found to be sensitive to the milieu of the polymer network in the following respects: (1) The esters were hydrolyzed faster in a chemically crosslinked polymer network than in soluble polymers at equivalent conditions; (2) Ester hydrolysis kinetics were dynamic during the gel-sol phase transition, the kobs decreased with the extent of hydrogel degradation; (3) a negatively charged polymer network inhibited the esters from being hydrolyzed in the polymer network in the hydrogel; and (4) solvent had a significant effect on ester hydrolysis kinetics, with the equivalent kobs in an H2 O environment about 1.8–2-fold greater than the NMRmeasured values in D2 O. Acknowledgment The authors gratefully acknowledge the Hong Kong General Research Fund (GRF16322016) and Science and Technology Plan of Shenzhen (JCY20170818114038319) for supporting the research work. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.actbio.2019.10.030. References [1] A. Vashist, Y.K. Gupta, S. Ahmad, Recent advances in hydrogel based drug delivery systems for the human body, J. Mater. Chem. B 2 (2) (2014) 147–166. [2] E. Caló, V.V. Khutoryanskiy, Biomedical applications of hydrogels: a review of patents and commercial products, Eur. Polym. J. 65 (2015) 252–267. [3] T. Vermonden, R. Censi, W.E. Hennink, Hydrogels for protein delivery, Chem. Rev. 112 (5) (2012) 2853–2888. [4] A.S. Hoffman, Hydrogels for biomedical applications, Adv. Drug Deliv. Rev. 54 (1) (2002) 3–12. [5] S.P. Zustiak, J.B. Leach, Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties, Biomacromolecules 11 (5) (2010) 1348–1357. [6] G.T. Chao, L.Y. Fan, Y. Lin, W.J. Jia, Z.Y. Qian, Y.C. Gu, C.B. Liu, X.P. Ni, J. Li, H.X. Deng, C.Y. Gong, M.L. Gou, K. Lei, A.L. Huang, C.H. Huang, J.L. Yang, B. Kan, M.J. Tu, Synthesis, characterization and hydrolytic degradation of degradable poly(butylene terephthalate)/poly(ethylene glycol) (PBT/PEG) copolymers, J. Mater. Sci. Mater. Med. 18 (3) (2007) 449–455. [7] C. Engineer, J. Parikh, A. Raval, Review on hydrolytic degradation behavior of biodegradable polymers from controlled drug delivery system, Trends Biomater. Artif. Organs 25 (2011) 79–85. [8] C. Hiemstra, L.J. Van Der Aa, Z. Zhong, P.J. Dijkstra, J. Feijen, Novel in situ forming, degradable dextran hydrogels by michael addition chemistry: synthesis, rheology, and degradation, Macromolecules 40 (2007) 1165–1173. [9] W.Van Dijk-Wolthuis, W.N.E. E.VanDijk-Wolthuis, J.A.M. M.Hoogeboom, M.J.V. anSteenbergen, S.K.Y.Y. Tsang, W.E. Hennink, Degradation and release behavior of dextran-based hydrogels, Macromolecules 9297 (97) (1997) 4639–4645. [10] W.N.E. Van Dijk-Wolthuis, O. Franssen, H. Talsma, M.J. Van Steenbergen, J.J. Kettenes-van den Bosch, W.E. Hennink, Synthesis, characterization, and polymerization of glycidyl methacrylate derivatized dextran, Macromolecules 28 (18) (1995) 6317–6322. [11] J.L. West, J.A. Hubbell, Polymeric biomaterials with degradation sites for proteases involved in cell migration, Macromolecules 32 (1) (1999) 241–244. [12] J.A. Burdick, C. Chung, X. Jia, M.A. Randolph, R. Langer, Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks, Biomacromolecules 6 (1) (2005) 386–391.
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