In Situ Forming and Dual-Crosslink Network Self-Healing Hydrogel

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In Situ Forming and Dual-Crosslink Network SelfHealing Hydrogel Enabled by a Bioorthogonal NopoldiolBenzoxaborolate Click Reaction with a Wide pH Range Di Wu, Wenda Wang, Diana Diaz-Dussan, Yi-Yang Peng, Yangjun Chen, Ravin Narain, and Dennis G Hall Chem. Mater., Just Accepted Manuscript • Publication Date (Web): 22 May 2019 Downloaded from http://pubs.acs.org on May 27, 2019

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Chemistry of Materials

In Situ Forming and Dual-Crosslink Network Self-Healing Hydrogel Enabled by a Bioorthogonal Nopoldiol-Benzoxaborolate Click Reaction with a Wide pH Range Di Wu,† Wenda Wang,‡ Diana Diaz-Dussan,‡ Yi-Yang Peng,‡ Yangjun Chen,‡ Ravin Narain, *, ‡ and Dennis G. Hall*, † †Department of Chemistry, ‡Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta T6G 2G2, Canada

ABSTRACT: The use of “click” chemistry as a hydrogel cross-linking reaction is often limited by slow reaction rates and harsh conditions such as exposure to UV light and/or use of non-specific or toxic reagents. On the other hand, the process of boronic ester formation between arylboronic acids and diols suffers from its intrinsic reversibility and low binding affinity at low pH, which impede its potential in many biomedical applications where a fast and stable “click” reaction is needed. Herein, we report a new concept of “click” hydrogel fabrication that combines a traditional sugar-based boronic ester and a novel nopoldiol-based benzoxaborolate as a dual-crosslink network (DCN) system. The cooperation of dynamic and rigid networks and the unique sensitivity of benzoxaborolate cross-links towards stimulus provide an intelligent hydrogel with a set of interesting features: (i) catalyst-/light-free nopoldiol-benzoxaborolate bioorthogonal “click” cross-linking; (ii) rapid in situ formation within 26 s; (iii) wide self-healing pH range from 8.5 to 1.5; (iv) exceptional stability under acidic condition and polyol solutions; (v) ROS-/pHresponsive degradation; (vi) pH-responsive drug release; and (vii) capability for viable cell encapsulation. The complementary “click” partners, a rigid diol monomer (1R)-(–)-nopol-methacrylamido-diol (nopoldiol) and a benzoxaborole-based monomer 5methacrylamido-1,2-benzoxaborole (MAAmBO), can be easily incorporated into a variety of synthetic polymers through freeradical polymerization with poly(ethylene glycol) methyl ether methacrylate (PEGMA) as the backbone component. The shortened gelation time, improved mechanical properties and excellent self-healing properties of the resulting DCN hydrogel PBNG were evaluated through rheological measurements; the stability/degradation of PBNG under low pH buffer and H2O2 were monitored via hydrogel weight changes; the potential of PBNG as a drug releasing carrier was assessed by pH-responsive release of doxorubicin (Dox) and, finally, HeLa cells were successfully encapsulated and cultured in the 3D network to confirm the hydrogel’s biocompatibility as cell culture scaffold. The non-toxic components and their fast “click” reaction under mild conditions make the nopoldiol-benzoxaborolate “click” hydrogels promising candidates for future biomedical applications such as gene delivery, cell therapy and tissue engineering.

INTRODUCTION Hydrogels are 3D cross-linked soft biomaterials that have been used widely in diverse biomedical applications such as controlled drug delivery, cell encapsulation and tissue engineering.1-4 Among the countless strategies to cross-link the 3D network structure, chemical ligations have afforded hydrogels with extensive versatility and intelligence. In the past decades, in situ forming hydrogels have attracted widespread attention in the biomedical community owing to their distinctive advantages such as minimally invasive implantation, which reduces patient’s discomfort, as well as ease of homogenous encapsulation of cells and bioactive molecules by mimicking their native extracellular matrix (ECM) microenvironment.5-6 More recently, “click” chemistry has emerged as a powerful and advantageous strategy for the in situ fabrication of hydrogels due to its high selectivity and specificity.7-16 Unfortunately, many common “click” reactions fail to meet the requirements of a suitable hydrogel cross-linking reaction such as bioorthogonality, use of safe reagents/triggers, formation of

benign side product, mild temperature and high reaction efficiency.17 For example, thiol-ene coupling reactions (Scheme 1A) require external photoinitiators and long UV exposure time, which could potentially damage cells and tissues;18-20 strain-promoted azide-alkyne cycloadditions (SPAAC) (Scheme 1B) are still restricted by slow reaction kinetics and a tedious synthesis of reactants;8, 21 Diels-Alder reactions (Scheme 1C) require high temperatures and long reaction times;22-23 tetrazines are sensitive to water and cellular thiols,24-25 and the tetrazine-norbornene inverseelectron demand Diels-Alder cycloaddition (IEDDA) (Scheme 1D) produces N2 as side product, which may ultimately affect the material’s mechanical integrity.10, 26 Despite all these efforts to eliminate external catalysis/triggers and to improve reaction rates, there is yet no ideal chemical ligation reaction for the preparation of bioorthogonal “click” hydrogels. To meet the rapidly growing demand for new and improved biomedical materials, hydrogels are desired that can exhibit

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fast self-healing and high stability to maintain their A) Thiol-ene

UV

+

SH

S

B) SPAAC N N N N

N N

+

C) Diels-Alder (DA) O +

O

N

O



O N

O

O

D) Tetrazine-norbornene IEDDA N N

– N2

+

N N

HN N

E) This work: tight nopoldiol-benzoxaborolate condensation O O + B OH

HO

HO

B O

O

Scheme 1. (A) – (D). Commonly used “click” reactions for in situ forming hydrogels. (E) New strategy based on tight benzoxaborolate formation for hydrogel cross-linking.

integrity during administration.27 It is notable, however, that most of the current self-healing hydrogels suffer from a slow healing process, limited healing conditions (pH, temperature or light) and unsatisfactory stimuli-responsiveness, which are desirable attributes in the development of biomaterials.28 Furthermore, because of the different pH conditions (1.0 ~ 8.0) of parts/tissues in the complex human body,29 hydrogels must be functional under a wide range of pH conditions to fulfill various applications such as localized oral drug release, targeted cancer drug delivery and wound dressing. To this end, dynamic covalent chemistry is emerging as a powerful tool to cross-link the hydrogel network with high efficiency and stability, while still providing the resulting hydrogels with self-healing behavior through a reversible reaction.28, 30 To date reversible boronate formation with cis-diols has become one of the most promising methods for the synthesis of biomaterials with excellent self-healing property (when pH > pKa of boronic acids) and unique pH-, glucose- and oxidative-responsiveness.31 However, the unfavorable binding of arylboronic acids with 1,2- or 1,3-diols under acidic pH, and the unspecific binding of boronic acids with endogenous polyols (glucose, fructose), greatly restrict the utility of boronate formation as an effective bioorthogonal “click” strategy in biomaterials. Numerous attempts have been made to improve the arylboronic acid/diol binding affinity by lowering the pKa of the boronic acid (e.g., with electron withdrawing substituents)32-34 or through stabilization of the resulting boronates with intra-/inter-molecular interactions (e.g. Wulff-type B–N coordination in o-aminomethylarylboronic acids).35-36 To this date, the lowest gelation pH of 4.0 for an

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arylboronic acid-based hydrogel was reported by Sumerlin and coworkers, which exploited intramolecular B–O coordination in 2-acrylamidophenylboronic acid (2APBA).37 Kiser and coworkers reported arylboronic acid/salicylhydroxamic acid-based hydrogel that forms at pH 4.2.38 An ideal boronate-based hydrogel that can tolerate both neutral and extreme pH conditions remains elusive. Benzoxaborole, a cyclic hemiboronic acid that has a lower pKa (7.2) compared to conventional arylboronic acids (8 ~ 9) and displays excellent binding affinity towards sugar- or catechol-based polymers under neutral and basic pH, has been studied by our group as a promising building block for hydrogels.39-43 Additionally, nopoldiol, a rigid unnatural cisdiol that provides fast, stable and bioorthogonal binding with arylboronic acids, was recently developed by our group for labeling cell surface and proteins.44-45 Herein, we report a novel type of dual-crosslink network (DCN) hydrogel involving benzoxaborolate formation with both sugar and nopoldiol to address the demands of modern hydrogels in regards to bioorthogonality, acid/polyol resistance, fast gelation, benign cross-linking reaction and by-products, and self-healing under a wide pH range. We hypothesized that the bioorthogonal “click” reaction between (1R)-(–)-nopolmethacrylamido-diol (nopoldiol) and 5-methacrylamido-1,2benzoxaborole (MAAmBO) moieties (Scheme 1E) would provide a rigid but slightly reversible network to ensure the stability of the hydrogel structure even under extremely acidic conditions and high concentration of polyols. Yet the highly labile and reversible sugar-benzoxaborolate crosslinks could preserve the hydrogel’s responsiveness by controlling the degree of cross-linking, thus, changing the pore size of the hydrogel upon stimulus. Moreover, our design affords dual-crosslink network (DCN) hydrogels through the fast ligation between three components embedded in two linear polymer chains without any requirement for heat, initiators, or UV light. Importantly, the benzoxaborolate formation generates water as the only byproduct, suggesting an ideal bioorthogonal and cytocompatible process for biomaterials. In this study, the binding affinity of MAAmBO with nopoldiol and a range of diols was studied, and the designed hydrogels were characterized by their gelation kinetics, mechanical strength, self-healing properties and stability. Furthermore, pH-responsive drug delivery using the DCN hydrogel PBNG was evaluated by releasing doxorubicin (Dox) as a model drug. Lastly, the suitability and biocompatibility of this “click” chemistry for cell and tissue engineering was confirmed by 3D encapsulation of HeLa cells.

RESULTS AND DISCUSSION Design and Preparation of Benzoxaborolate-based Hydrogels. To investigate the roles of both dynamic and rigid benzoxaborolate networks, a series of PEG precursors containing different compositions of MAAmBO, nopoldiol and 2-gluconamidoethyl methacrylamide (GAEMA) were synthesized through a facile and efficient free-radical polymerization with a targeted degree of polymerization (DP) of 200. The desired composition of each components could be easily obtained (Table 1) without any post-

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Figure 1. Graphic illustration of the design and preparation of benzoxaborolate-based single and dual-crosslink network (DCN) hydrogels.

Table 1. Composition of polymers. Polymer Composition (mol%)a PEGMA

GAEMA

nopoldiol

MAAmBO

PB

87.0

–

–

13.0

PNG

75.2

17.2

7.6

–

PG

83.3

16.7

–

–

PN

92.5

–

7.5

–

PN’

82.3

–

17.7

–

aCalculated

from

1H

NMR signal integration.

functionalization. The statistical benzoxaborole- or diolembedded copolymers were characterized by 1H NMR spectroscopy and GPC, and the detailed results are shown in Table1 and Table S1. Poly(ethylene glycol) methyl ether methacrylate (PEGMA) was chosen as the backbone to ensure the hydrophilicity and biocompatibility of the hydrogels, and polymer PB was designed with a fixed molar ratio of 13% to allow sufficient binding with GAEMA and nopoldiol in the diol-containing polymers. Polymers PN and PG, which contained similar molar percentages of nopoldiol and GAEMA as polymer PNG, were synthesized to demonstrate the roles and properties of each component in the resulting hydrogels. Polymer PN’ with 17.7 mol% of nopoldiol was designed to compare with PG, which contained similar proportions of GAEMA moieties (16.7 mol%). As illustrated in Figure 1, the hydrogels formed by mixing PB with PNG, PN, PN’ and PG were denoted accordingly as PBNG, PBN, PBN’ and PBG, where only PBNG is a DCN hydrogel. The latter three are single network (SN) hydrogels prepared for control studies. Solgel transition from polymer precursors to hydrogel PBNG under neutral pH occurred rapidly within seconds by gentle swirling of the solution mixture (Figure 1 and Video S1).

Studies of MAAmBO – Diol Benzoxaborolate Formation Under pH 8.5 to 1.5. To demonstrate the superior binding ability of benzoxaborole with nopoldiol under a wide range of pH conditions, several diols that are commonly used for boronate formation in biomaterials

preparation were studied for comparison. Since a higher pH is usually preferable for benzoxaborolate formation,46-47 greater conversions to the benzoxaborolate complex at high pH were observed in all cases (Table 2). Remarkably, nopoldiol, a rigid cyclic cis-diol, provides an extremely stable benzoxaborolate with the MAAmBO monomer even at pH 1.5 (59.5% conversion). The unusual hydrolytic stability of this benzoxaborolate is attributed to the entropy barrier48 and the steric bulkiness of the nopoldiol, which slows down the dissociation process by hindering the approach of water. Similarly, 3-methylacrylamido phenylboronic acid (MAPBA), a traditional phenylboronic acid, also formed nopoldiol-boronate with high conversion under a wide range of pH (see Supporting Information); however, the resulting boronate was not as dynamic as nopoldiol-benzoxaborolate (entry 2), which limits its use in self-healing, pH-responsive, and biodegradable materials. On the other hand, 2-lactobionaidoethyl methacrylamide (LAEMA), GAEMA and catechol, which are the most popular diol partners for arylboronic acids in the design of stimuli-responsive biomaterials for gene therapy,49-51 biomolecular recognition52 and tissue scaffold,40 showed moderate binding affinity towards MAAmBO at pH 7.4 (about 60% conversion), and only trace amount of the benzoxaborolate complex was observed even at the slightly acidic pH of 5.2. Moreover, biological polyols (D-glucose, D-fructose) and the ribose-containing anti-cancer drug (capecitabine), demonstrated low to moderate binding affinity with MAAmBO under basic/neutral condition and very poor affinity under acidic environment. Hydrogel formation tests also support that only nopoldiol-containing polymers can form stable hydrogels with benzoxaborole/boronic acid-containing polymers (PB or PB’) under a wide range of pH (Figure S1). These results confirm the unique tightness and acid-resistance of the nopoldiolbenzoxaborolate complex. Table 2. Results of benzoxaborolate conversions between MAAmBO and various diols

3



Entry

Diols

Conversion to benzoxaborolate %a pH 8.5

pH 7.4

pH 5.2

pH 1.5

1

nopoldiol

86.5

79.6

65.5

59.5

2

nopoldiolb

88.2

86.9

84.7

79.9

3

LAEMA

78.2

61.5

9.29

–

4

GAEMA

82.4

64.7

7.38

–

5

D-glucose

29.3

9.90

–

–

6

D-fructose

73.0

51.7

6.33

–

7

catechol

77.4

57.3

9.45

–

8

capecitabine

57.7

38.1

2.91

–

aThe

conversion to benzoxaborolate (%) of mixing MAAmBO and diols was determined by 1H NMR. bConversion to boronate (%) of mixing MAPBA and nopoldiol (see Supporting Information for details).

To further characterize the benzoxaborolate formation reaction between MAAmBO and nopoldiol, 1H NMR studies were performed under buffered conditions at pH ranging from 8.5 to 1.5. The successful formation of nopoldiol-benzoxaborolate was observed at all of the pH conditions. Interestingly, under acidic pH condition, the downfield effect of selected proton signals on the benzoxaborolates suggests the ring-opening of the oxaborole moiety on the nopoldiol-benzoxaborolate 1 to give the neutral nopoldiol-benzoxaborolate 2 (Scheme 2). Benzoxaborole ring opening is normally unfavorable;53 however, in this case, the low pH favors the neutral boronate and the very rigid diol unit is Scheme 2. Equilibrium formation of nopoldiolbenzoxaborolate complex (1 and 2) under general and acidic conditions.

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MAAmBO alone. This significant change in chemical shift indicates a strong coordination between the two molecules (MAAmBO and nopoldiol) and the sp3 character of the boron in the nopoldiol-benzoxaborolate 1. Whereas, the boron chemical shift of the neutral benzoxaborolate 2 under lower pH conditions is identical to the MAAmBO alone, which corroborates its predominant sp2 character of the boron in the nopoldiol-benzoxaborolate 2. Furthermore, the formation of 1 and 2 was observed by ESI-MS analysis (Figure S5), where the signals of both complexes (465.2 m/z) were detected at pH 7.4 and 1.5. Notably, at pH 1.5, no degradation of the benzoxaborolate, such as protodeboronation, was observed by LC-MS (Figure S6). These results support the expectation that the formation of nopoldiol-benzoxaborolate is clean and tight under a wide range of pH conditions.

Rheological Measurements of Hydrogels on Gelation Kinetics, Mechanical Strength and SelfHealing Properties. With the encouraging molecular details of monomer complexation in hand, mechanical properties of DCN and SN hydrogels were characterized through a series of dynamic rheological measurements. Molar ratios of diols (nopoldiol and GAEMA) against the benzoxaborole partner (MAAmBO) in the abovementioned hydrogels were calculated (Table 3) to allow a better understanding of the function of each component. Firstly, the gelation time was de determined via the measurements of modulus versus time. Specifically, storage modulus G' and loss modulus G'' were monitored after mixing 10 w/v% of two polymer solutions (PB and PNG/PG/PN/PN’), and the gelation time was defined when the storage modulus G' surpasses loss modulus G''. As shown in Table 3 and Figure 3A, the DCN hydrogel PBNG displayed the fastest gelation process of 26 s (T1), which was 7 ~ 8 times shorter than hydrogels with either GAEMA or nopoldiol alone (T2 and T3). Furthermore, nopoldiol was observed to play a more important role, since the gelation process was greatly accelerated by increasing the nopoldiol content (T3 vs T4), and with the similar molar ratios of reactants, the gelation kinetics of PBN’ (T4) was significantly higher than that of PBG (T2). The advantages of benzoxaborole over conventional arylboronic acids were further demonstrated by replacing MAAmBO in polymer PB with MAPBA (polymer is denoted as PB’, Figure 2) and assessing their binding rate with diol-based polymers via gelation time measurement (Figure S7 and Table 3). As expected, MAPBA, with a relatively higher pKa (~ 9), showed lower reactivity towards boronate formation with diol monomers, thus, resulting in a 2 ~ 10 Figure 2. Structures of polymer PB and PB’.

even less likely to open compared to the o-hydroxymethyl arm of MAAmBO. This hypothesis is confirmed by 11B NMR chemical shifts (Figure S4), where the boron signal of the nopoldiol-benzoxaborolate at basic and neutral pH has an obvious upfield shift of about 12 ppm compared to the

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Figure 3. Rheological measurements of hydrogels and macroscopic demonstration of hydrogel self-healing properties. (A) Hydrogel gelation process. (B) Dynamic oscillatory frequency sweeps of hydrogels in 10 w/v%. (C) Dynamic oscillatory frequency sweeps of 10 w/v% PBNG at pH 7.4 to 1.5. (D) Oscillatory strain sweep, (E) step-strain test and (F) demonstration of self-healing property of 10 w/v% PBNG at pH 7.4.

fold slower gelation process in comparison with the corresponding MAAmBO-based hydrogels. Altogether, these results reveal that both nopoldiol and MAAmBO are key reactive components to achieve a fast gelation that is crucial for in situ forming hydrogels. To determine the mechanical strength of different hydrogels and evaluate the influence of pH, dynamic oscillatory frequency sweep measurements were performed. As shown in Figure 3B, among the four different hydrogels,

PBNG with the largest total molar ratios of reactive components, exhibited the highest storage modulus G'. Due to the lower binding affinity between MAAmBO and GAEMA components, hydrogel PBG with a high sugar molar ratio of 1.27, was much weaker than PBN (containing only 0.55 molar ratio of nopoldiol). The influence of pH conditions on the mechanical strength was also tested on DCN hydrogel

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Table 3. Summary of molar ratios of reactive components in hydrogels and the resulting gelation time. Entry

Molar ratiob

Hydrogela

Gelation time, T (s)c

MAAmBO

MAPBA

nopoldiol

GAEMA

0.60

1.36

26

1

PBNG

1

–

2

PBG

1

–

–

1.27

204

3

PBN

1

–

0.55

–

230

4

PBN’

1

–

1.37

–

79

5

PB’NG

–

1

0.57

1.29

276

6

PB’G

–

1

–

1.21

427

7

PB’N

–

1

0.53

–

1114

8

PB’N’

–

1

1.30

–

211

aAll

the hydrogels are formed with10 w/v% solid content at pH 7.4. PBNG (10 w/v%) at pH 7.4, 5.2 and 1.5 buffers (Figure 3C). The G' (at 𝛾 = 1%, 𝜔 = 1 Hz) of PBNG dropped drastically from 3896 Pa at pH 7.4 to 886.4 Pa and 747.6 Pa at acidic pH (5.2 and 1.5, respectively), indicating the cleavage of the dynamic sugar-benzoxaborolate cross-links and possibly some of the nopoldiol-benzoxaborolate crosslinks under acidic condition. This phenomenon is consistent with the above study of monomer reactivity (Table 1), where nearly all of the GAEMA benzoxaborolate complex was released at pH 5.2, whereas, only about 15% of the acidresistant nopoldiol-benzoxaborolates were dissociated at the same pH. Additionally, SEM images further confirmed the formation of porous networks in DCN and SN hydrogels, as well as PBNG at acidic pH (Figure 4). As expected, hydrogels with higher G' and pH value exhibit smaller pore sizes and more compact network structures, corroborating their stronger mechanical properties.

bCalculated

1H

cStrain

by NMR. 1%, frequency 1 Hz. and hydrogel types (Figure 3B and 3C). Here, the DCN hydrogel PBNG (10 w/v%) was selected to demonstrate the self-healing properties in a wide range of pH (8.5 to 1.5). For instance, PBNG (10 w/v%) was tested, at pH 7.4, with linear amplitude sweep tests ( 𝛾 = 1% to 1000%) to determine the linear viscoelastic region (Figure 3D). Out of the plateau, the storage modulus G' dramatically dropped at the critical strain of 334%, indicating collapse of the gel network. Afterwards, strain sweep tests with 𝛾 = 1% and 400% were applied cyclically on the hydrogel to test its ability for modulus recovery upon gel failure (Figure 3E). Satisfactorily, in all cases, nearly quantitative storage modulus G' were recovered over two cycles (Figure 3E, Figure S8A – D and S9A – D), demonstrating the excellent self-healing ability of PBNG under all ranges of body pH conditions. It is notable that, without the help of dynamic sugar-benzoxaborolates, PBN is HN +

O

3

O

HN +

HO HO

nopoldiol

HO

O H2O2

1a

nopoldiol

HN

Unlike permanently cross-linked networks formed by covalent bonds, which usually exhibit constant G' and G'', all of the designed hydrogels showed frequency-dependent storage modulus G'' and loss modulus G'', regardless of pH

HN O B O O

HO HO

B OH

Figure 4. SEM image of hydrogels. (A) PBNG at pH 7.4, (B) PBN at pH 7.4, (C) PBG at pH 7.4, (D) PBNG at pH 1.5. Hydrogels were all made in 10 w/v% and freeze-dried 16 h after gelation. Scale bars are equal to 20 𝜇m.

O

B O

O

OH

+ O

+

B(OH)3

OH 5 4

Scheme 3. Equilibrium and oxidative degradation of nopoldiol-benzoxaborolate complex (1a). also self-healing with good efficiency at pH 7.4 (Figure S8E and Figure S9E). These results indicate that, although the nopoldiol-benzoxaborolate cross-links are considered rigid, both diol-benzoxaborolate cross-links show a certain degree of dynamicity that contributes to the excellent self-healing properties of the resulting hydrogel. Moreover, the selfhealing ability of hydrogel PBNG was also demonstrated macroscopically by connecting two hydrogel cubes (Dox loaded and non-loaded), followed by 20 seconds of healing without any external forces (Figure 3F). The reconstituted

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Figure 5. Stability studies under conditions of H2O2, biological polyols and pH stress. (A) H2O2-Responsive degradation profile of hydrogel PBNG. (B) Stability of hydrogels PBNG, PBG and PBN under polyol solution of 30 mM D-glucose and 15 mM D-fructose. (C), (D) Hydrogel stabilities of PBNG and PBN, respectively, at pH 7.4 to1.5.

hydrogel could be lifted manually and was found to tolerate its own weight, indicating the success of fracture selfrepairing. Stability Studies Under Conditions of H2O2, Biological Polyols and pH Stress. A series of stability tests of DCN and SN hydrogels were planned by systematic exposure to oxidants, polyols and various pH. Given that the microenvironment of tumor and many other pathological conditions are highly associated with the overexpression of reactive oxygen species (ROS), benzoxaborolate crosslinked hydrogel networks are very appealing for biomedical applications such as ROS-responsive local delivery of therapeutic agents, and for the antioxidative regulation of overproduced ROS.54-55 Herein, ROS-responsive degradation of hydrogels was investigated by using different concentrations of hydrogen peroxide as ROS models. As expected, a higher concentration of H2O2 resulted in faster degradation, and longer exposure times contributed to more complete degradation (Figure 5A). Furthermore, the oxidation process was modeled using monomers in a model reaction. As depicted in Scheme 3 and the Supporting Information, compound 3 (benzoxaborole) was used as a model of MAAmBO to bind nopoldiol, and the degradation process was monitored by 1H NMR. By adding 1, 5 and 10 mM of H2O2 to a mixture of 3 and nopoldiol monomer (1:1 molar ratio, 5 mM of each). The conversion to benzoxaborolate complex (1a) was found to decrease from the initial level of 63% to 57%, 36% and 22%, respectively, after 20 min. The amount of oxidation product of 3 and 1a, 2-hydroxybenzyl alcohol (5), was found to increase over

time, meanwhile, nopoldiol was released as a free diol or as a boric acid-conjugate (4). These results further confirm the high efficiency of benzoxaborolate oxidative cleavage, and the ROS-responsive degradation properties of the resulting hydrogels. Subsequently, hydrogel stability against polyol solutions was investigated to assess the bioorthogonality of this system towards endogenous sugars. Notably, in contrast to other boronate-based hydrogels, nopoldiol-benzoxaborolate hydrogels are resistant to high concentration of sugars. As shown in Figure 5B, SN hydrogel PBG with only sugarbenzoxaborolate cross-links degraded quickly over 120 min, while PBNG and PBN could maintain their weight. Additionally, nopoldiol-benzoxaborolate complex 1 (Scheme 2) was subjected to a high concentration of competing Dglucose, and 1H NMR monitoring confirmed the extraordinary stability of this complex (Figure S12). Remarkably, 30 mM of D-glucose, which is about four times the level in diabetic human blood stream, did not affect the equilibrium conversion of monomer MAAmBO and nopoldiol to nopoldiol-benzoxaborolate 1 at pH 7.4 (79.0 mol% and 81.0 mol% with and without D-glucose, respectively). Due to the high bioorthogonality of the DCN hydrogel PBNG, it could be of potential use in diabetic wound healing as well as sustainable insulin/drug delivery. To evaluate the acid stability swelling and degradation behaviors of the hydrogels, 10 w/v% PBNG and PBN were immersed in pH buffers of 7.4, 5.2 and 1.5, and their weights were monitored. Both hydrogels were able to maintain their

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Figure 6. pH-Responsive doxorubicin drug release. (A) Cumulative Dox release profile of hydrogel PBNG (10 w/v%) at pH 7.4 to 1.5. (B) Picture of hydrogels at pH 7.4 to 1.5 after 48 hours of drug releasing. (C) Graphic description of DN hydrogel PBNG pH-responsive drug release.

weight over several days under acidic pH (Figure 5C and 5D); however, PBNG exhibited twice the lifetime (~20 days) of PBN (~10 days). The higher stability of PBNG could be attributed to its higher cross-link density. It is notable that, unlike other permanent/irreversible bonds formed by “click” reactions, the nopoldiol-benzoxaborolate cross-links can slowly degrade by hydrolysis, especially under acidic pH. The pH-dependency of nopoldiolbenzoxaborolate formation was also indicated through the monomer studies previously shown in Table 1, where 86.5% and 59.5% of benzoxaborolate conversions were obtained under pH 8.5 and 1.5, respectively. Moreover, unlike PBN, the PBNG hydrogel showed a pH-dependent swelling rate (Figure 5C vs 5D); a highly desirable feature for controlled drug release. This behavior suggests that the highly pHsensitive sugar-benzoxaborolate cross-links tends to break down faster at lower pH, consequently, the enlarged porous gel structure could allow more water uptake and subsequent release of the encapsulated cargo. pH-Responsive Drug Release Study. The aforementioned results of pH-dependent swelling behavior of PBNG, along with its tolerance to low pH environment hold promise for the application of this DCN hydrogel in sustainable and pH-controlled drug release. The pHresponsive drug release capability of hydrogel PBNG was verified by encapsulating and releasing doxorubicin (Dox), a widely used anti-cancer drug. Distinctive differences in the drug releasing rate at pH 7.4, 5.2 and 1.5 are displayed in the cumulative drug release profile (Figure 6A). After 8 hours, a significant amount of Dox (68.5%) was released at pH 1.5 compared to 49.2% and 21.8% at a pH 5.2 and 7.4, respectively. Plateaus were reached after 32 hours, and the cumulative release totaled 89.8%, 79.3% and 37.5% at pH of

1.5, 5.2 and 7.4, respectively, after 48 hours. Distinguishable color changes were also observed for the hydrogel after 48 h (Figure 6B). The hydrogel at pH 1.5 showed the lightest color from the unreleased drug residue, and the redness increased for the gels exposed at higher pH. As demonstrated in Figure 6C, both sugar- and nopoldiolbenzoxaborolates formed stable networks under neutral pH, where drugs can be efficiently encapsulated inside the compact double cross-linked network. However, once the hydrogels are exposed to an acidic environment (such as that of tumor site, or gastric acid), the rigid nopoldiolbenzoxaborolates are able to maintain the integrity of the hydrogel for about 20 days (showed previously in Figure5C), while the acid-sensitive sugar-benzoxaborolates dissociate rapidly, thus releasing the drug upon gel swelling. Overall, its slow degradation under acidic conditions, combined with a sustainable pH-controlled drug release confirm the potential of the DCN hydrogel a biodegradable and “smart” carrier for targeted drug delivery. Cytotoxicity and 3D Cell Encapsulation. With a view to exploit the biological potential of the DCN hydrogel, MTT cytotoxicity assay was planned to evaluate the biocompatibility of individual polymers PB and PNG. To this end, HeLa cells were incubated with polymer solutions of 0.01~1 mg/mL for 24 hours, and nearly 100% of cell viability in polymer PB and PNG solutions were observed in all cases except for highly concentrated (1 mg/mL) PNG (Figure 7A). Furthermore, cytotoxicity of the hydrogel was also measured by incubating HeLa cells with hydrogel extracts, which were obtained 24 and 48 hours after immersing the hydrogels into cell culture medium with different hydrogel/medium weight

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Figure 7. Cytotoxicity assays and 3D cell encapsulation images. MTT assay of (A) individual polymers (PB and PNG), and (B) hydrogel extract of PBNG. (C) 2D and 3D images of Live/Dead assay of HeLa cells cultured in PBNG (10 w/v%) for 24 and 48 hours. (D) Cell viability of the encapsulated cells (measure with confocal microscope images).

ratios. Over 95% of cells were found viable after 24 hours, while slightly lower viabilities (over 86%) were observed after 48 hours (Figure 7B), which could result from the leaching of unbound or degraded polymers over this longer period. Nevertheless, no significant toxicity was found either in the free polymers or hydrogel extracts, which further demonstrates the excellent biocompatibility of the designed materials. Since hydrogels bear many similarities with native tissues, such as a porous structure and a highly hydrated scaffold, they are regarded as one of the most promising soft materials for extracellular matrix (ECM).56 Therefore, we set out to further explore the spontaneous 3D cell encapsulation ability of PBNG (optimal hydrogel) by mixing the two polymer precursors with HeLa cells. Gelation occurred rapidly within seconds, and the cells were distributed homogeneously in the 3D structure, followed by 24 and 48 hours of incubation under standard cell culture environment. A live/dead assay was utilized to differentiate the live (green) and dead (red) cells, and confocal microscopy was applied to image the cells in the hydrogel in both 2D and 3D imaging (Figure 7C). As quantified in Figure 7D, 93% and 83% of cells were alive after 24 and 48 hours respectively, thus, revealing the hydrogel’s ability to transport nutrients efficiently through the porous structure and to provide a microenvironment conducive of maintaining cellular function.

An in situ forming and dual-crosslink network (DCN) hydrogel, PBNG, was designed based on two distinct benzoxaborolate cross-links. The combination of dynamic sugar-benzoxaborolate cross-links and rigid nopoldiolbenzoxaborolate cross-links allows the PBNG hydrogel to become self-healing across an exceptionally wide range of pH (8.5 to 1.5), with unusually high tolerance to acid. Furthermore, the nopoldiol-benzoxaborolate bioorthogonal “click” chemistry endows the desired hydrogel with many attributes such as an ultra-fast gelation rate (< 26 s), benign cross-linking chemistry (light/catalyst-free, water is the only by-product), ROS-responsive degradation and good resistance towards low pH conditions and biological polyols. The DCN hydrogel exhibits pH-responsive swelling capabilities that contributes to its efficient pH-controlled release of small molecules, as exemplified with the anticancer drug doxorubicin (Dox). Moreover, the promising biomedical potential of the DCN hydrogel PBNG for cell therapy and tissue engineering is supported by cytotoxicity assays, as well as successful 3D encapsulation and culture of HeLa cells. As novel and unique “click” partners, MAAmBO and nopoldiol components could be easily incorporated in diverse polymeric hydrogels to achieve bioorthogonality, acid resistance, ROS-responsiveness, facilitated gelation, and enhanced mechanical properties that could fulfill specific biomedical requirements.

CONCLUSIONS

ASSOCIATED CONTENT Supporting Information.

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The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Experimental details; chemical composition and molecular weights of polymers; spectroscopic data for compounds, binding studies, oxidation studies and polyol competition studies; additional rheological data; procedures of drug release study; procedures of cell toxicity tests and 3D cell encapsulation.

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected]

ORCID Ravin Narain: 0000-0003-0947-9719 Dennis G. Hall: 0000-0001-8555-6400 Diana Diaz-Dussan: 0000-0003-1778-3964 Yangjun Chen: 0000-0002-7449-9348

ACKNOWLEDGMENT This work was funded by the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Canada Foundation for Innovation (CFI) and the University of Alberta. We thank Prof. Hongbo Zeng (rheometer), Mr. Gareth Lambkin (cell culture techniques and equipment), nanoFAB Faculty (FESEM imaging) and Imaging Faculty Cross Cancer Institute (confocal laser scanning microscope) for their help and comments.

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In Situ Forming and Dual-Crosslink Network Self-Healing Hydrogel

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