Molecular Engineering Mechanically Programmable Hydrogels with

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Molecular Engineering Mechanically Programmable Hydrogels with Orthogonal Functionalization Lizhu Wang, Liang Zhu, Michael Hickner, and Baojun Bai Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.7b03398 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 13, 2017

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Chemistry of Materials

Molecular Engineering Mechanically Programmable Hydrogels with Orthogonal Functionalization Lizhu Wang,*† Liang, Zhu,‡ Michael Hickner,‡ Baojun Bai*† †

Department of Chemical Engineering, Missouri University of Science and Technology, Rolla,

MO 65409, USA ‡

Department of Materials Science and Engineering, The Pennsylvania State University,

University Park, PA 16802 USA *To whom correspondence should be addressed. E-mail: [email protected], [email protected]

Abstract A unique orthogonally tunable synthetic polymer hydrogel with programmable elasticity is described herein. The temporal modulation of mechanical properties was achieved by an orthogonal sulfonium-containing synthon, acting both as crosslinks and as functionalizable handles for chemical modification of the gel. The kinetic formation of in-situ covalent crosslinked hydrogels with ionic features enabled time-dependent mechanical behavior over weeks, a critical parameter of biomimetic substrates for cell development. The elasticity of the dynamic hydrogel was approximately two orders of magnitude greater than the ionically crosslinked sample with constant stiffness. In addition, we achieved on-demand control of the elastic properties of the hydrogels by application of a thermal stimulus of 37 °C, which provides new avenues to regulate cell behavior and fate. Furthermore, sulfonium groups and styrenyl moieties within the network provided covalent attachment sites for molecules of interest via highly efficient nucleophilic substitution and thiol-ene chemistry. This robust and orthogonal strategy has been demonstrated for temporal control of elasticity and molecular functionalization of the hydrogels as potential substrates for cell development. 1 ACS Paragon Plus Environment


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Introduction

Synthetic hydrogels, 3-dimensional highly-hydrated macromolecular networks, are of great interest for applications in tissue engineering, drug delivery, as barrier materials and in biosensors.1,2 The applications of hydrogels in biomaterials and regenerative medicine require mechanical integrity and the ability to introduce chemically active moieties as substrates to facilitate cell growth. In many biological scenarios of embryonic development and tumorigenesis, matrix stiffness in the cellular microenvironment is highly dynamic over a broad range of timescales. Engler, et al.3-5 has demonstrated that the engineered elasticity of the substrates was capable of directing cell behavior. The increased elasticity of the hydrogels monotonically enhanced the growth of human mesenchymal stem cells.6 Thiol functionalized natural hyaluronic acid crosslinked with diacrylate poly(ethylene glycol) generated dynamic hydrogels with temporal stiffness changes due to the continuous Michael addition of thiol and acrylate, whose elasticity was consistent with heart muscle development. 7-10 The time-dependent mechanical properties of the hydrogels were able to regulate and enhance physiological processes of cell proliferation and production of collagen. Moreover, the hydrogels with temporally modulated mechanical stiffness facilitated stem cell maturation by mimicking their native environment.11 These reported results highlight the importance of hydrogel elasticity on cell response. Temporal control of elasticity in hydrogels over weeks could potentially afford optimal substrates by recapitulating in vivo microenvironments.12 Hydrogels based on biopolymers of hyaluronic acid and alginate, an essential component of extracellular matrix for mediating morphogenesis and cellular signaling, have been used for tissue engineering due to their tunable stiffness and facile functionalization.13-16 Thus, there is a need to develop

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temporally mechanical modulation of synthetic polymer-based hydrogels with tunable functionality to tailor cell substrates for specific applications. Ionically physical hydrogels based on dynamic cationic and anionic bonds demonstrated multiple functionalities of mechanical toughness and self-healing in physiological conditions having biocompatibility property.17 In particular, one-pot free radical polymerization of anionic monomers in the presence of equimolar cationic moieties generated polyampholyte hydrogels while two-step sequential polymerization of ionic monomers produced polyion complex hydrogels. The weak ionic bonds distributed in these gels offer unique features of toughness, self-healing and adhesive properties.18, 19 However, polyion complexes based hydrogels prepared by traditional strategies cannot achieve time-dependent mechanical manipulation in a programmable fashion, where the mechanical properties remain constant.20 Finally, the absence of chemically active groups in these materials renders the hydrogel unavailable for further functionalization. It is therefore desirable to expand current synthetic approaches of polyion complexes based hydrogels to achieve hydrogels with temporally tunable elasticity. In the development of topological polymer chemistry with programmed macromolecular chemical structures, a variety of techniques have been implemented to facilitate single ring and multi-cyclic preparation. Electrostatic self-assembly and covalent fixation (ESA-CF) approaches have been deployed to generate well-defined topological polymers in which non-covalent polymeric structures were constructed from carboxylates containing telechelic polymer precursors and cyclic ammonium or sulfonium molecules via electrostatic interaction and converted to covalently linked products. As an example, telechelic polymers having cyclic sulfonium or ammonium end moieties along with carboxylate counteranions for quantitative conversion to the corresponding ester bridges through nucleophilic attack upon heating have



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been demonstrated. Tezuka, et al.21 created a wealth of topological polymers using ESA-CF in combination with selectively covalent linking approaches including metathesis condensation and click chemistry. In addition, treatment of deprotonated carboxy groups on a nylon surface with pyrrolidinium compounds generated a highly hydrophobic surface with a contact angle greater than 130°.22-25 A 5-membered cyclic sulfonium tethered to benzyl groups was found to be sufficiently stable, which allowed the incorporation of molecules by nucleophilic reaction of sulfonium cations.26 Furthermore, the orthogonal monomers containing nucleophilic active sulfonium sites and radically active styrenyl moieties were applied to synthesize block copolymers for chemical modification, introducing a variety of useful chemical motifs.27-33 However, electrostatic self-assembly and covalent fixation has never been applied to hydrogel synthesis due to synthetic challenges of incorporating the correct functional units into a crosslinked matrix though secondary covalent crosslinks were introduced to poly(ethylene glycol) and hyaluronic acid hydrogels.34,

35

We envision that the introduction of sulfonium

moieties to radically active polymerizable monomers could provide an avenue to fabricate the ionically crosslinked hydrogels containing kinetically controlled in-situ covalent bridges induced by heating. Inspired by the ionic hydrogels formed by sequential polymerization of ionic monomers and ESA-CF chemistry, we sought to prepare mechanically programmable hydrogels from sulfonium containing orthogonal synthons. Herein, we demonstrated how the mechanical properties of the synthesized hydrogels are temporally programmed through our orthogonal monomer approach by unique tunable dynamic ionic bonds and covalent fixation. The presence of cationic moieties and orthogonally polymerizable monomers enables us to prepare ionic hydrogels and kinetically convert ionic linkages to in-situ covalent bridges, leading to


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dynamically tunable hydrogels over broad timescales. The orthogonal monomer provides chemical handles for functionalization by exploiting the pendant sulfonium sites and radically active styrenyl groups. This concept offers the advantages of temporal mechanical manipulation by virtue of in-situ formation of kinetically controlled covalent crosslinks and the incorporation of bioactive molecules on the chemical handles of the orthogonal monomers for biologicalappropriate materials.

Experimental

Materials. All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) except as noted. Tetrahydrothiophene (THT), vinylbenzyl chloride (VBC), 2-mercaptoethanol (BME), potassium persulfate (KPS), 2,2'-azobis(2-methylpropionamidine) dihydrochloride (V50) and sodium thiosulfate pentahydrate (STS) were used as received. Acrylic acid (AA) and 2acrylamido-2-methylpropanesulfonic

acid

(AMPS)

were

neutralized

to

generate

the

corresponding sodium salt (Na-AA and Na-AMPS). (4-vinylbenzyl)tetrahydrothiophenium chloride (VBTht) and (4-vinylbenzyl)triethylammonium chloride (VBTEA) were prepared according to previous literature.36,37

Orthogonal VBTht and Cationic VBTEA Monomers Synthesis. To a stirring solution of VBC (18.24 g, 120 mmol) in methanol (20 mL) was dropwise added THT (16 g, 180 mmol) at room temperature and stirred for 4 d in the dark. The solution was concentrated under reduced pressure and the residual was triturated with hexane, filtered, dried in vacuo at room temperature to afford a white solid (24 g, Yield: 83 %). 1H NMR (400 MHz, DMSO-d6) δ 7.56 (m, 4H), 6.77 (dd, J = 18.0, 11.2 Hz, 1H), 5.91 (d, J = 17.6, 1H), 5.35 (d, J = 10.8 Hz 1H), 4.57 (s, 2H), 3.43 (m, 4H),



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2.19 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 138.3, 135.9, 130.8, 129.4, 127.0, 115.8, 44.8, 42.6, 28.2. VBTEA was synthesized in a similar fashion of VBTht. The structural characterization was consistent with the reported data37 (refer to Figure S1 in supporting information). 1H NMR (400 MHz, DMSO-d6) δ 7.60 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 6.79 (dd, J = 18.0, 11.2 Hz, 1H), 5.95 (d, J = 17.6, 1H) 5.35 (d, J = 10.8 Hz, 1H), 4.74 (s, 2H), 3.16 (q, J = 7.2 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 138.8, 135.9, 132.8, 127.4, 126.6, 116.0, 59.3, 52.0, 7.7.

2-acrylamido-2-methylpropanesulfonic Acid Sodium Salt (Na-AMPS). To a stirring solution of AMPS (20.7 g, 100 mmol) in ethanol (200 mL) was added solid NaOH (4 g, 100 mmol) at room temperature. The reaction mixture was vigorously stirred for 1 h and Na-AMPS precipitated from ethanol. The product was filtered, washed with ethanol (20 mL x 3) and dried in vacuo at room temperature to give a white solid (21 g, Yield: 92 %). Sodium acrylate (Na-AA) was prepared in a similar fashion to Na-AMPS.

Synthesis of AA-AMPS Copolymers. The anionic copolymers were synthesized in aqueous media. A typical polymerization is as follows: to a stirring solution of Na-AA (0.24 g, 25 mmol), Na-AMPS (10.88 g, 47.5 mmol) and V-50 (13.6 mg, 0.05 mmol) in DI water (75 mL) was bubbled with argon for 30 min and immersed in a preheated oil bath at 70 °C until the solution reached high viscosity. The reaction solution was quenched by liquid nitrogen for 5 min and warmed to room temperature over 30 min. The reaction mixture was precipitated three times in a large amount of acetone and then dried in vacuo at 50 °C to yield copolymer of P(AA-AMPS) as a white solid which was used without further purification.


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Synthesis of Mechanically Programmable Hydrogels. All hydrogels syntheses were carried out in a similar fashion in glass vials. The hydrogels were prepared by polymerization of cationic monomers in the presence of anionic copolymers. A typical hydrogel polymerization is as follows: to a stirring solution of AA-AMPS (1 g, Na-AA, 0.23 mmol) in DI water (6 mL) at 0 °C was added VBTht (1.2 g, 5 mmol), bubbled with argon for 30 min followed by addition of KPS (3.2 mg, 0.012 mmol) and STS (3.6 mg, 0.015 mmol) and kept in a refrigerator at 0 °C for 12 h. During this process, the ionic crosslinked hydrogel was formed. The ionic crosslinked sample was termed as IA5-V100 based on molar fraction of Na-AA (~ 5 mol%) in AA-APMS copolymer and VBTht (100 mol%) monomer percentage relative to repeat units in AA-AMPS during hydrogel preparation. Thermal treatment of the as-prepared ionic hydrogel at 80 °C generated covalent crosslinks in the sample, termed as CA5-V100 (Refer to Table S1 for detailed samples information in supporting information). The resulting hydrogels were immersed in DI water for further characterization and modification. The addition of cationic VBTEA monomers during hydrogel preparation introduced hetero-ionic crosslinks within the hydrogels.



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Results and discussion

Scheme 1. General synthetic scheme of mechanically programmable hydrogels via sequential polymerization from anionic and orthogonally cationic monomers. The asterisk (*) represented sulfonium cations in the hydrogel for further covalent attachments by nucleophiles.

Synthesis of Mechanically Programmable Hydrogels. The mechanically tunable hydrogels in a temporal fashion were prepared by the formation of polyion complexes via sequential polymerization of ionic monomers followed by covalent fixation as borrowed from topology chemistry as shown in Scheme 1. More explicitly, the hydrogels were fabricated from water soluble

P(AA-r-AMPS)

anionic

copolymers

in

the

presence

of

cationic

4-vinyl-

benzyltetrahydrothiophenium chloride (VBTht) and (4-vinylbenzyl)triethylammonium chloride (VBTEA) monomers under redox radical conditions as elaborated in this work.


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Figure 1. 1H (400 MHz) and 13C (100 MHz) NMR spectra of orthogonal monomer VBTht in DMSO-d6.

The cyclic sulfonium VBTht monomer was isolated by direct alkylation of tetrahydrothiophene with vinylbenzyl chloride in methanol in excellent yield using a method similar to that reported in previous literature.36 A styryl group capable of radical polymerization and a cyclic cationic sulfonium motif providing ionic and covalent crosslinks coexist in the monomer with orthogonal functionality. The chemically active styryl and sulfonium groups also offered functionalizable sites by thiol-ene chemistry and nucleophilic substitution as demonstrated in this research. Furthermore, the excellent water solubility of chloride form VBTht facilitated the hydrogel synthesis. The cyclic sulfonium moieties maintained chemical stability and were not vulnerable to weakly nucleophilic sulfonate counterions under our synthetic conditions (refer to Figure S2 in supporting information). This chemical stability ensured the integrity of the cyclic sulfonium moiety during the hydrogel synthesis.



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The orthogonal monomer VBTht was the critical pivot in the temporal hydrogel, governing the mechanical tunability of the material. The structural assignment of VBTht was based on its 1H and 13C NMR spectra as shown in Figure 1. The appearance of a quartet peak at 4.56 ppm (-PhCH2-) is consistent with the formation of 4-vinylbenzyl chloride sulfonium salt while the signals at δ Hb, 6.77 (d, J = 18.0, 11.2 Hz, 1H) and Ha , 6.00 (d, J = 17.6 Hz, 1H) ppm in the olefinic region of the 1H NMR corresponded to the two protons in the vinyl group.36 In particular, the signals at δ 3.43 (m, 4H) and 2.19 (m, 4H) ppm in the 1H NMR were assigned to cyclic sulfonium protons. The ratio of cyclic sulfonium protons (PhCH2) (3.43 ppm) to benzyl protons (-PhCH2-) (4.56 ppm) was maintained at 2:1 as displayed in Figure 1. These observations along with the 13C NMR spectrum supported the formation of the VBTht sulfonium salt.

Figure 2. The formation of covalent crosslinks from carboxylate nucleophilic substitution to sulfonium moieties.

The preparation of the orthogonal hydrogels is illustrated in Scheme 1 by redox polymerization via the formation of polyion complexes.38 Polymerization of VBTht cationic monomers in the presence of P(AA-r-AMPS) anionic copolymers generated ionically crosslinked polyelectrolyte hydrogels, which were mechanically soft and stretchable due to the


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entanglement of ionic polymer chains. Hetero-ionic crosslinks were introduced into the gel by the cationic VBTEA monomer to finely tune the mechanical properties of the material. Thermal treatment of the ionic crosslinked hydrogels induced the formation of the double crosslinked polymeric network with ionic and crosslinkable covalent features. In-situ generation of the covalent crosslinks resulted from the nucleophilic attack of the chemically sensitive cyclic sulfonium cations by carboxylate (COO-) to form the ester bridges at temperatures greater than 0 °C as shown in Figure 2. The characteristic peak at 5.04 ppm in the 1H NMR spectrum corresponding to a benzyl ester (-PhCH2OCO-) supported the formation of covalent crosslinks (see Figure S3 in supporting information). This strategy is similar to the process of covalent fixation through electrostatic self-assembly, where a myriad of topological polymers have been designed by ring-opening cyclic sulfonium moieties by nucleophilic carboxylates (COO-).39 Although the nucleophilic reactivity of the carboxylate anion in the presence of the sulfonium counteranion is significantly reduced in aqueous media due to the hydration of the anion,40 in our case, the nucleophilic substitution between carboxylates and sulfonium ions occurred inside the hydrogels even at room temperature as demonstrated by dynamic moduli increase (vide infra). Controlling the ratio of cationic VBTht to the anionic moieties in the ionic copolymers rendered sulfonium and carboxylate additional reactive motifs of sulfonium and carboxylates in the hydrated gel for further chemical functionalization with molecules of interest. This approach offers new opportunities to fabricate hydrogels containing bioactive molecules for cell development.



(A)

(B) 5

IA5-V100

IA20-V100 IA10-V100

G', G" (Pa)

10

4

10 G', G'' (Pa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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CA20-V100

CA10-V100 4

10

CA5-V100

3

10

0

20

40

60

80 100 120 140 160 Time (s)

0

20

40

60

80 100 120 140 160 Time (s)

Figure 3. Time sweep profiles of shear elastic moduli G' (filled symbols) and viscous moduli G" (open symbols) at 25 °C, (A) as-prepared ionic hydrogels formed by sequential polymerization of ionic monomers, IA5-V100, IA10-V100 and IA20-V100 made from 5, 10, 20 mol% AA containing P(AA-AMPS) anionic copolymers, respectively; (B) double crosslinked hydrogels induced by temperature, CA5-V100, CA10-V100 and CA20-V100 prepared from the corresponding precursors of IA5-V100, IA10-V100 and IA20-V100 upon thermal treatment at 80 °C for 6 h in water.

Mechanical Properties of Programmable Hydrogels. The mechanical properties of hydrogels are of great importance as biologically appropriate materials for immature cell growth, which influence proliferation, differentiation, migration and adhesion of the cells.41,42 In this study, the shear elastic modulus, G', a quantitative metric of substrate stiffness, were measured as a function of time at a fixed frequency of 1 Hz. As indicated in Figure 3, the introduction of cationic VBTht monomers to anionic P(AA-r-AMPS) aqueous solution generated soft ionic hydrogels via polyion complexes while thermal treatment of the ionic hydrogels produced tough 12 ACS Paragon Plus Environment

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hydrogels with ionic and covalent crosslinks. The shear elastic moduli of all the samples were always greater than the viscous moduli at the time scale of our measurements, showing a significant elastic response. The ionically crosslinked hydrogels using VBTht had similar shear elastic moduli, ranging from 4.2 to 5.0 KPa, ascribed to their similar density of ionic crosslinks, which was reflected in their consistent water uptakes of 250 ± 20 wt%. The elastic moduli of the ionically crosslinked samples were in the same range of the polyion complex (5.9 KPa) prepared using cationic VBTEA and Na-AMPS comonomers under similar conditions. This also demonstrated the ionic feature of the single cationic VBTht based samples where the sulfonium moieties remained intact to some extent. However, after higher temperature treatment, the hydrogels showed a substantial increase in elastic response due to the formation of the covalent crosslinks from nucleophilic substitution. As exhibited in Figure 3, the shear elastic moduli increase also implied the generation of a chemically crosslinked network in the samples after thermal exposure. More interestingly, the covalently crosslinked sample CA20-V100 showed more than one order of magnitude higher shear elastic modulus of 76.6 KPa compared to the ionically crosslinked sample IA20-V100 having a shear elastic modulus of 5.0 KPa, suggesting the formation of chemical crosslinks via orthogonal monomers through covalent fixation. The elasticity enhancement is ascribed to the crosslinked network by the locally covalent interactions of polymer chains within the hydrogels. The mechanical behaviors of the hydrogels at biological temperature of 37 °C would be described (vide infra) to accommodate such materials for cell culture. In addition, Our synthetic hydrogels could be ideal candidates for further development as cell substrates by virtue of their elastic properties as reported in stem cell lineage commitment where osteogenesis occurred in the elastic range of 11-30 KPa and adipogenesis dominated in soft microenvironments of 2.5-5 KPa.43,44 By tuning cationic sulfonium moieties and carboxylate



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groups, we could generate substrates with a wide range of mechanical properties from 5 to 76.6 KPa for cell development. The self-stiffening behavior rendered such materials being applied as temperature induced reinforcing medical implants for bone regeration and craniofacial implants.45

Figure 4. Dynamic shear elastic moduli G' of the as-prepared ionic crosslinked hydrogels of IA5-V100 of A, B and IA20-V100 of C, D and IAM-VBTE of E as a function of time upon standing in water at biologically relevant temperature of 37 °C and room temperature of 25 °C, respectively, for 4 weeks. Sample IAM-VBTE was prepared from anionic PAMPS and cationic VBTEA monomers under similar conditions to hydrogels of IA5-V100 and IA20-V100. The images on the right were the hydrogels after 4 weeks.

The time-dependent shear elastic moduli, an assessment of the relative stiffness of the hydrogel, were monitored as a critical parameter in the design of biomimetic materials. Dynamic tuning of hydrogel elasticity was demonstrated by treating the hydrogels at room temperature and performing rheometry after standing in water. The hydrogels in this work had similar 14 ACS Paragon Plus Environment

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physical appearance, Figure 4. However, they showed radically different mechanical behavior. The elasticity of the hydrogels having sulfonium and carboxylate moieties gradually increased with time over weeks as shown in Figure 4 while the polyelectrolyte hydrogel IAM-VBTE had a constant mechanical response over time due to the absence of the formation of in-situ covalent bridges. For instance, the shear elastic modulus of hydrogel IA5-V100 increased by 3-fold from 5 to 14.6 KPa while sample IA20-V100 had approximately one order of magnitude increase in elastic modulus (G' = 50.1 KPa) compared to its initial mechanical strength (G' = 5 KPa) at room temperature. However, under biologically relevant conditions, the elasticity of IA20-V100 sample increased to ~ 400 KPa. The increase in the carboxylate concentration within the hydrogels and treatment temperature dramatically improved the stiffness as the number of in-situ covalent bridges increased, generating a greater overall change of in modulus. However, the hydrogels cured at 37 °C showed decreased swelling ratio of 150 ± 50. Synthetic VBTht based hydrogels showed long-term elastic modulation over 4 weeks on a continuous basis without an external stimulus such as UV irradiation. Burdick et al. reported in-situ stiffened methacrylated biopolymers from physically crosslinked hydrogels, where UV (365 nm) was employed to produce the secondary polymerization without adverse effect on human mesenchymal stem cells.4



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B

A

Figure 5. SEM images of hydrogel IA20-V100 (A) as-prepared sample, (B) standing at room temperature for 30 days.

Furthermore, the pore size of the hydrogel in dry state decreased upon standing as shown in Figure 5. The reduction in pore size indicated the formation of covalent crosslinks within the hydrogels. These data are in agreement with substantially dynamic modulus increase in hydrated measurements. In previous research, the stiffness of alginate hydrogels was modulated by intermittent near-infrared triggered release of calcium (Ca2+) on a non-continuous basis.7 The natural hyaluronic acid hydrogels had a narrow range of apparent modulus increase over less than 1 week.3 The unique approach here enabled temporally controlled hydrogel stiffness spanning soft, intermediate and the relatively stiff substrates by tuning carboxylate concentration over time periods greater than weeks. The range of elasticity for our hydrogels spanned the differential stem cell lineage commitment as reported in previous work (2.5 – 30 KPa).8 We anticipate that the time-dependent mechanical properties by tuning cationic sulfonium moieties and anionic carboxylate groups allow the construction of kinetically stiffened hydrogels. The synthetic polymer based hydrogel displayed temporal elasticity over weeks, which could mimic


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the stiffness of the matrix in their native conditions. The results were similar to previous work on in-situ stiffening of poly(ethylene glycol) based hydrogels by a second thiol-ene polymerization for directing valvular interstitial cell phenotype.15,46 Temporal modulation of hydrogel elasticity to regulate cell behavior will be evaluated both in vitro and in vivo experiments in our future work.

The thermal history of the hydrogels has been demonstrated to influence their stiffness by promoting nucleophilic covalent crosslinking. Surprisingly, the shear elastic modulus of hydrogel CA20-V100 dramatically increased to 27.3 MPa from 5 KPa upon standing in water at room temperature for 1 month, which was about two orders of magnitude greater than the asprepared ionically crosslinked sample IA20-V100 as indicated in Figure S4 (see supporting information). In stark contrast, we found the sample cured at 0 °C for 30 days had a much lower shear elastic modulus of 0.03 MPa under which the sulfonium cations are believe to be less chemically active toward nucleophilic substitution by carboxylate (COO-). From our observations, we can infer that the elasticity increase of the hydrogel resulted from the increased covalent crosslinks of carboxylates and sulfonium motifs by covalent fixation. From an engineering standpoint thermal treatment can be harnessed to mechanically tune synthetic hydrogels in-situ, generating stiff materials from soft hydrogels. Generally, the hydrogel stiffness in the presence of water is reduced due to the plasticization effect. The behavior of the hydrogel is similar to mechanically morphing materials, whose mechanical strength is responsive to an external cue as found the increased stifness in sea cucumbers under threatened conditions. The unique property of the hydrogels could be employed for construction of active damping systems, intelligent robotics and machanosensory equipment.47, 48



100

7 (B)

80

CA20-V100

60

40

20 CA5-V100

0

5

CA10-V100

20

10

Elastic Modulus G' (KPa)

(A) Elastic Modulus G' (KPa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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6 5

IA20-V100

4 3 2

IA20-V80

1 0

IA20-V60 100

Carboxylate Fraction (%)

80

60

Sulfonium Fraction (%)

Figure 6. Dynamic shear elastic moduli G' of the prepared hydrogels of CA5-V100, CA10-V100 and CA20-V100 after thermal treatment at 80 °C for 6 h in water. Sample IA20-V80 and IA20V60 was synthesized from 20 mol% AA containing P(AA-AMPS) anionic copolymers under similar conditions to hydrogel IA20-V100. The elasticity was measured in its pristine state.

Temporal control of matrix stiffness was demonstrated to determine the biological relevance of these materials during which the extracellular matrix undergoes dynamic changes over time. In our study, we could achieve on-demand control of the elastic properties of the hydrogels by an on-demand stimulus, which would provide new avenues to regulate cell behavior and cell fate. The hydrogel elasticity increased with carboxylate moieties upon thermal treatment. The hydrogels consisting of 5, 10 and 20 mol% -COO- in the anionic copolymers had shear elastic moduli of 12.0, 16.2 and 76.6 KPa, respectively, as revealed in Figure 6A. Apparently, the on-demand elasticity of the hydrogels can be tuned by the concentration of


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carboxylate groups upon thermal treatment. Furthermore, the hydrogels become more compliant by decreasing the concentration of sulfonium motifs. As displayed in Figure 6B, the initial stiffness of the hydrogels can be softened from a shear elastic modulus of 5 to 0.8 KPa as the sulfonium fraction relative to anionic moieties decreased. Thus, our approach has high fidelity for tunability of the hydrogel elasticity to model physiological systems by varying the concentration of ionic moieties and the temperature of thermal treatment before seeding of cells. This synthetic route is suitable for tuning the hydrogels within the range of soft to stiff tissues.

5

10

(A)

(B)

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3

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10

IA10-V100 3

10

1

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0.1

1 10 Strain (γ %)

100

0

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10 -1 ω (rad s )

2

10

Figure 7. Rheological data of the hydrogels, (A) strain amplitude sweeps (γ = 0.1 – 100 %); (B) frequency sweeps (ω = 0.1-100 rad s-1) of the samples.

As shown in Figure 7A, strain amplitude sweeps of the hydrogels demonstrated an elastic behavior. The shear elastic moduli of the hydrogels markedly decreased over the critical regimes, indicative of a collapse of the gel form to a quasi-liquid state. The linear viscoelastic regime of the ionically crosslinked hydrogel (< 9 %) was less than that of the crosslinked counterparts with covalent features (30 – 90 %). In our case, the increase in elasticity was due to in-situ formation of covalent bridges. Furthermore, the rheological properties of the synthesized 19 ACS Paragon Plus Environment


hydrogels were further studied as a function of angular frequency. As indicated in Figure 7B, the elastic behavior (G' > G") was prevalent over the entire frequency range, suggesting solid-like behavior. However, the double crosslinked hydrogels showed ionic character as observed from their moduli’s dependence on angular frequency while covalent polyacrylamide hydrogels displayed frequency-independent shear elastic moduli.49

CA10-V100

G', G" (Pa)

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4

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0

200

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600

800

1000

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Figure 8. Step strain measurement of the hydrogel CA10-V100 on a continuous basis (three cycles).

Interestingly, these samples exhibited rapid recovery of the mechanical properties after a large-amplitude oscillatory breakdown, termed thixotropy. When subjected to a large-amplitude oscillation (γ = 200 %, ω= 6.28 rad s-1 (1 Hz)), the G' decreased from 5 to 1 KPa, in a quasiliquid state (tan δ = 2). However, when the amplitude was subsequently decreased to a small value (γ = 1 %) within the linear viscoelastic regime at a frequency of 1 Hz, the G' immediately recovered to its initial value of 5 KPa and the network returned to quasi-solid state (tan δ = 0.7). As shown in Figure 8, the recovery was reproducible for at least three cycles. The rheological data implied the in-situ crosslinked sample with ionic characteristics is also an excellent 20 ACS Paragon Plus Environment

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candidate as a fast-recovering hydrogel for soft tissues where the hydrogels constantly experienced rapid deformation and release. The recovery behavior is similar to cationic oligomeric gelator containing hydrogels as reported by Yoshida, et al., but the G' of 14.4 KPa is one magnitude higher than that of polypeptide copolymer based hydrogels containing ammonium side chains.50

Scheme 2. Direct coupling and post-functionalization by thiol-ene chemistry and nucleophilic addition, (A) the introduction of 2-mercaptoethanol by monomer approach; (B) the modification through the reaction of the residual active moieties with of 2-mercaptoethanol.

Orthogonal Functionalization of the Hydrogels. Direct functionalization is a convenient method for hydrogel modification to encapsulate bioactive molecules. The orthogonal nature of the sulfonium monomer enabled functionalization sites for thiol-ene chemistry and nucleophilic substitution. Direct thiol-ene functionalization involved the chemical coupling of thiol21 ACS Paragon Plus Environment


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containing biomolecules and the styrenyl sulfonium monomer. As demonstrated in our work in Scheme 2, a test vehicle of 2-mercaptoethanol was introduced to VBTht monomers through thiol-ene chemistry (see Figure S5 and S6 in supporting information). The presence of cationic sulfonium moieties facilitated the encapsulation of the model BME molecule by increasing its aqueous solubility.

To broaden the scope of the hydrogel functionalization, an indirect functionalization route that would allow the attachment of a wide range of molecules within the network under mild conditions was designed using residual functional groups. The post-functionalization strategy takes advantage of the ease of chemical handling in the nucleophilic reaction of cyclic sulfonium to a variety of nucleophiles of thiol, primary amine and selenol containing molecules, enabled by the residual sulfonium moieties in the hydrogels.26 More explicitly, commercially available 2-mercaptoethanol as test case was applied to modify the hydrogels with cyclic sulfonium sites for post-incorporation of functional molecules. The nucleophilic conjugation reaction was demonstrated by the corresponding model reaction of 2-mercaptoethanol with VBTht under aqueous media. Furthermore, the successful conjugation of 2-mercaptoethanol to the hydrogels was validated by FT-IR spectroscopy (see Figure S7 in supporting information). The addition of functionalized molecules during hydrogel synthesis can produce covalent attachment of molecules of interest through orthogonal functionalization in a well-defined hydrogel matrix, which could serve as substrates for cell culture. We can envision that cysteine modified Ac-GCGYG-RGDSPG-NH2 (RGD) adhesive peptide could be incorporated in the hydrogel matrix via thiol-ene chemistry or nucleophilic conjugation to create functional moieties in film form for cell adhesion following the established protocols.51


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Conclusions

In conclusion, the temporal mechanical modulation of hydrogel elasticity has been described. The control of the mechanical strength of the hydrogels has been demonstrated by inducing the ion-containing hydrogels to ionic and covalent crosslinks upon thermal treatment. The kinetically controlled formation of the covalent crosslinks from sulfonium cations and carboxylate moieties under aqueous conditions manipulated the time-dependent mechanical properties of the materials over weeks. The temporal modulation of hydrogel elasticity could mimic the stiffness of the substrates in their native micro-environments. The nucleophilic susceptible sulfonium sites within the network offered functionalizable motifs by introducing molecules of interest to functionalize the gel. This study illustrates the importance of an orthogonal monomer containing radical active moieties and nucleophilic reactive sites for the design of biological and mechanically morphing materials. The orthogonal moieties allowed the chemical handling of the hydrogels. Thus, the new concept in this study provides an avenue to build temporal modulation of hydrogel elasticity and modulus, which occurs in biological contexts.

Associated Content

Supporting Information. Detailed characterization data of the hydrogels is provided including monomers preparation, hydrogel synthesis and functionalization of hydrogels and their characterizations.

Author Information Corresponding author. *Email: [email protected],[email protected]



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Acknowledgements

Funding for this project is provided by Missouri University of Science and Technology.

References

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Molecular Engineering Mechanically Programmable Hydrogels with Orthogonal Functionalization Lizhu Wang,*† Liang, Zhu,‡ Michael Hickner,‡ Baojun Bai*† †

Department of Chemical Engineering, Missouri University of Science and Technology, Rolla,

MO 65409, USA ‡

Department of Materials Science and Engineering, The Pennsylvania State University,

University Park, PA 16802 USA Table of Contents Graph


Molecular Engineering Mechanically Programmable Hydrogels with

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