Letter pubs.acs.org/macroletters
Photoregulated Hydrazone-Based Hydrogel Formation for Biochemically Patterning 3D Cellular Microenvironments Malar A. Azagarsamy,†,§ Ian A. Marozas,†,§ Sergio Spaans,‡ and Kristi S. Anseth*,† †
Department of Chemical and Biological Engineering, the Howard Hughes Medical Institute and the BioFrontiers Institute, 596 UCB, University of Colorado at Boulder, Boulder, Colorado 80303, United States ‡ Department of Biomedical Engineering, Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, Netherlands S Supporting Information *
ABSTRACT: Photodriven click reactions have emerged as versatile tools for biomaterial synthesis that can recapitulate critical spatial and temporal changes of extracellular matrix (ECM) microenvironments in vitro. In this article, we report on the synthesis of poly(ethylene glycol) (PEG) hydrogels using photodriven step-growth polymerization, where one of the reactive functionalities is formed by a photocleavage reaction. Upon photocleavage, an aldehyde functionality is generated that rapidly reacts with hydrazinefunctionalized PEGs; the gelation kinetics and final material modulus are distinctly controlled by variations in the light intensity. This light-driven aldehyde generation is further exploited to install biochemical ligands in the hydrazone-based hydrogels with precise spatial control. We expect that userdirected spatial and temporal control over both biophysical and biochemical gel properties through photochemical reactions and photopatterning, respectively, should provide newfound opportunities to probe and understand dynamic cell−matrix interactions.
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synthetic hydrogels for cell encapsulation.10 The photoclick reactions, such as the thiol−ene reaction, are particularly interesting, as these allow further control of the spatialtemporal chemistry of synthetic ECM environments. For example, Fairbanks et al.14 demonstrated that the thiol−ene photochemistry could be utilized not only to fabricate hydrogel scaffolds but also to site-specifically incorporate peptide cues, such as the cell-adhesive RGD sequence and the cell-degradable KCGPQGIWGQCK sequence. These reactions allowed for cellular encapsulation and the creation of hydrogel environments to observe and study cell adhesion, proliferation, and migration in real time. In a similar manner, the thiol−ene photochemical reaction has also been used as an orthogonal reaction to the copper-free SPAAC chemistry to spatiotemporally incorporate biochemical moieties in SPAAC-produced hydrogel scaffolds.9 While these are elegant approaches to fabricate hydrogels with spatiotemporally varying properties, thiol−ene photochemical reactions typically employ radical photoinitiators, which can sometimes compromise the biological integrity of proteins, such as growth factors, that are often desired to control cell growth, differentiation, and secretory properties.
ynthetic hydrogels, fabricated from well-defined synthetic polymeric precursors, have emerged as powerful cell culture platforms for recapitulating some of the complex and dynamic aspects of the extracellular matrix (ECM) in vitro.1−3 Specifically, these synthetic platforms have allowed researchers to precisely tune cellular microenvironments in three dimensions by providing well-defined mechanical and biochemical niches to study the impact of matrix signaling on various cell functions, including adhesion, migration, proliferation, and differentiation.4−7 However, synthetic hydrogels engineered for such advanced biological studies need to meet a set of criteria: (i) polymer gelation must occur under physiological conditions, particularly with respect to temperature and pH and (ii) the chemistry employed for gelation and subsequent biochemical incorporations must be efficient, bioorthogonal, and cytocompatible, as all of these reactions are performed in the presence of cells. Over the past few years, bioorthogonal click chemistry has been widely employed as a versatile cross-linking tool to form polymeric hydrogels, particularly those used for cell encapsulation, due to their quick, efficient, and cytocompatible chemistries and reaction conditions.8−10 To date, numerous bio-orthogonal click chemistries, including strain-promoted azide−alkyne cycloaddition (SPAAC),9 oxime ligation,11 Michael addition,12,13 photoinitiated thiol−ene chemistry,14 and tetrazine−norbornene cycloaddition,15 have been employed to fabricate poly(ethylene glycol) (PEG) based © XXXX American Chemical Society
Received: September 18, 2015 Accepted: December 6, 2015
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DOI: 10.1021/acsmacrolett.5b00682 ACS Macro Lett. 2016, 5, 19−23
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ACS Macro Letters
Figure 1. Photodriven hydrazone-based PEG hydrogel formation. (a) Molecular structures of 4-arm PEG macromers: PEG hydrazine 1 and PEG nitrobenzyl 2. 10 wt % hydrogels were formed in PBS at 25 °C and pH 7.5 upon exposing equimolar concentrations of 1 and 2 (5 mM, 5 wt %) to 365 nm light wavelength at 20 mW/cm2. (b) Photoinduced formation of the 2-nitrosobenzaldehyde from 2-nitrobenzyl alcohol and immediate capturing of the aldehyde by hydrazine to form the hydrazone bond. (c) Evolution of the storage modulus (G′) with time when a solution of 1 and 2 was exposed to 365 nm light wavelength at 20 mW/cm2. Inset indicates the crossover point between G′ and G″.
Scheme 1. Synthesis of PEG Macromers (10 kDa): 4-Arm PEG Hydrazine 1 and 4-Arm PEG Nitrobenzyl 2 That Are Utilized as Precursors for Photodriven Hydrazone Hydrogel Formation
More recently, hydrazone- or oxime-based hydrogel scaffolds that are formed by the condensation of hydrazine or hydroxylamine with aldehyde were reported for cellular encapsulations.11,16,17 As these hydrazone/oxime bonds are reversible in nature, McKinnon et al. further demonstrated that hydrazone-based covalently adaptable networks possess viscoelastic and dynamic properties similar to soft tissues and also that the reversibility of the bond can be tuned to allow for the proliferation and spreading of C2C12 myoblasts and axon extension from mouse embryonic stem cell derived neurons.16,18 We speculated that if the hydrazone hydrogel formation could be driven by light it would allow precise external control over: (i) gel formation kinetics and (ii) spatiotemporal incorporation of biochemical ligands. Driven by this rationale, we developed a photocontrollable approach to fabricate step-growth PEG hydrogels that are cross-linked via reversible hydrazone bonds by the condensation of aldehyde and hydrazine. Here, the photoresponsiveness
is introduced by incorporating a photoreactive 2-nitrobenzylalcohol moiety, which forms a 2-nitrosobenzaldehyde (Figure 1) when exposed to UV−visible light (365−405 nm).19 We leverage this in situ photogenerated aldehyde and its reaction with hydrazine to produce hydrazone cross-linked PEG hydrogels. Experiments were conducted to quantify the gel formation kinetics as a function of light intensity and pH, which enabled us to achieve varied final gel moduli. We further demonstrated that the light-driven hydrazone chemistry can be employed to spatiotemporally incorporate specific biochemical ligands within the hydrogel scaffold. The structures of the PEG-based macromolecular precursors employed in the hydrogel synthesis are provided in Figure 1. As shown in Scheme 1, 4-arm hydrazine-terminated PEG (10 kDa), PEG-Hydrazine 1, was obtained by coupling tri-Bochydrazinoacetic acid to 4-arm PEG tetra-amine and then deprotecting the Boc moieties under acidic conditions (see SI for complete synthetic details). Similarly, 4-armed PEG20
DOI: 10.1021/acsmacrolett.5b00682 ACS Macro Lett. 2016, 5, 19−23
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ACS Macro Letters
intensities, i.e., for 10 and 5 mW/cm2. Specifically, the polymeric solutions exposed to 5 and 10 mW/cm2 took ∼1270 and ∼880 s, respectively, to reach 90% of the final modulus, while 20 and 30 mW/cm2 took only ∼550 s. Further, only a minimal difference in the rate of gelation was observed for polymeric mixtures exposed to 20 and 30 mW/cm2 (Figure 2a). Interestingly, as can be gleaned from Figure 2a, a clear variation in the storage modulus at ∼15 min (900 s) was seen for polymeric mixtures exposed to varied light intensities, suggesting that this light-controlled strategy could also be leveraged to control the storage modulus with external light intensities and without the need for structural variations or change in solution composition. Temporal control over the mechanical properties of this system was also explored by dosing the precursor solution to 365 nm light at an intensity of 30 mW/cm2 for intervals of 2 min. Indeed, polymerization only occurred at times of light exposure, and the G′ remained constant when not dosed with light (Figure 2b). Thus, the mechanical properties of this system can be fine-tuned based upon the intensity and duration of light exposure. Importantly, this system possesses advantages over reports of hydrogel scaffolds with independent mechanical properties and ligand densities;25,26 if polymerized off stoichiometry with an excess of hydrazine for ligand incorporation, this scaffold is capable of in situ spatiotemporal control over matrix stiffness independent of ligand density and without the need for swelling in a crosslinking agent postpolymerization. Interestingly, we observed that PEG-nitrobenzyl 2 is capable of homopolymerization in the absence of PEG-hydrazine 1. However, the time scale for the onset and completion of gelation for this reaction is almost 3-fold slower than the copolymerization of PEG-NB and PEGHY, even at 10 wt % PEG-NB (see SI for details), which suggests that the capture of photogenerated aldehydes by hydrazine is extremely rapid compared to other side reactions. We expect that over 95% of the aldehydes were consumed based upon the rheological data, but future work will explore this further to quantify and understand the aldehyde consumption by hydrazone condensation. Next, the effect of pH on the kinetics of hydrogel formation was evaluated since pH is known to significantly affect hydrazone bond formation.21,22 Here, the gelation kinetics were studied at three different pH conditions: acidic, neutral, and basic. For these experiments, the pH of the final solutions of the macromolecular precursors in PBS was adjusted to 6.0, 7.5, and 8.5. The polymeric precursors 1 and 2 were then mixed and exposed to 20 mW/cm 2 of 365 nm light. The polymerization proceeded most rapidly at a neutral pH of 7.5 compared to both acidic and basic conditions (Figure 2c). We reason that the increased polymerization rate at neutral pH is due to the utilization of an aliphatic hydrazine, which is relatively more nucleophilic than the acyl hydrazide or phenyl hydrazine used in other reported hydrazone-based hydrogels22,23 and bioconjugation systems.24 Furthermore, this enhanced gelation rate at neutral pH was also recently observed by McKinnon et al.,17 when hydrazine was used to form hydrogel networks. Interestingly, we also observed the highest final storage modulus for gelation at neutral pH, as compared to either the basic or acidic conditions (Figure 2d), suggesting that the neutral state may provide conditions that lead to the formation of a more ideal network. We then investigated the possibility of sequential photodriven reactions: first forming the hydrogel using light-triggered hydrazone formation and then spatiotemporally photopattern-
nitrobenzyl−OH (10 kDa) 2 was obtained upon coupling PEG tetra-amine to 2-nitrobenzyl-butanoic acid 3, which was synthesized following similar routes of a previously reported synthetic procedure.20 Hydrogel formation was evaluated under aqueous conditions at 25 °C using rheological studies. Specifically, a 1:1 stoichiometric solution of the 4-arm PEG macromers 1 and 2 at a final density of 10 wt % in PBS (each macromer at 5 mM, 5 wt %) was exposed to collimated 365 nm light at 20 mW/cm2. Upon light exposure, a rapid increase in the storage modulus (G′) was observed (Figure 1b), indicating hydrazone bond formation between PEG 1 and 2 and evolution of the network structure, as result of the light-induced generation of aldehyde species in 2 and subsequent capturing of the resultant aldehydes by super nucleophilic hydrazines of 1. The crossover between G′ and the loss modulus (G″), which is an estimate of the gel point,9 was noted after 70 ± 5 s. The time to 90% of the final plateau modulus was less than 10 min, and the resulting final storage modulus was ∼15 kPa. The gels remained stable in aqueous conditions at 37 °C for up to 5 days. Both the reaction time, time to reach the gel point, and final network properties suggest the formation of a covalently cross-linked hydrogel under conditions that are facile for cell encapsulation. A great benefit of light-driven reactions is that their rates can be finely tuned in situ and on demand by varying the amount of photons delivered. To quantify the ability to externally control the kinetics of gel formation, the light intensity was varied from 5 to 30 mW/cm2. As shown in Figure 2a, faster network formation is observed when the solution of polymeric precursors 1 and 2 is exposed to higher light intensities, 20 and 30 mW/cm2, but slower gel formation was seen at lower
Figure 2. Effect of light intensity and duration on gelation kinetics: (a) storage modulus evolution as a function of time for 5 mW/cm2, 10 mW/cm2, 20 mW/cm2, and 30 mW/cm2 intensities of 365 nm light exposure at pH = 6.0. (b) Storage modulus evolution in response to 2 min doses of 30 mW/cm2 light. Polymerization only occurs at times of light exposure (shaded boxes). (c) Hydrogel storage modulus evolution as a function of time with 20 mW/cm2 at different pH conditions: pH = 6.0, pH = 7.5, and pH = 8.5. (d) Corresponding storage modulus at 90% of final modulus at different pH values (n = 3). 21
DOI: 10.1021/acsmacrolett.5b00682 ACS Macro Lett. 2016, 5, 19−23
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Figure 3. Photopatterning of biochemical peptides. (a) Schematic representation of the sequential approach to first form off-stoichiometric hydrazone hydrogel and then photopattern peptides. (b) Structure of fluorescein-labeled 2-nitrobenzyl alcohol-functionalized RGD peptide: NBK(fluorescein)GRGDS. (c) Fluorescent image of patterned fluorescein-labeled RGD peptide in hydrazone hydrogel via photodriven hydrazone formation chemistry. NB-K(fluorescein)GRGDS is swollen into a off-stoichiometric (1:0.5) hydrazone hydrogel and irradiated through a chrome photomask with 365 nm light at 20 mW/cm2 for 5 min (scale bar: 250 μm). (d) Corresponding 3D image indicating the immobilization of RGD throughout the hydrogel with 200 μm depth (scale bar: 250 μm).
resulting hydrogels formed with tethered RGD peptide, NBKGRGDS (1 mM, Figure 3b), for cellular adhesion. After encapsulation, the hMSCs were cultured under standard culture conditions (see SI for details) for 120 h, and Live/Dead staining was used to assess cell viability at 24, 48, and 120 h. Images were quantified using confocal microscopy, and results showed 90 ± 4%, 82 ± 6%, and 78 ± 8% cell viability at 24, 48, and 120 h postencapsulation, respectively (Figure 4). Although
ing specific biochemical ligands as pendant functional groups within the hydrogel scaffold using the same chemistry (Figure 3a), such that there should be negligible changes in the local mechanical properties of the network. Specifically, a fluorescein-labeled cell adhesive RGD peptide 14 NB-K(fluorescein)GRGDS was synthesized using solid-phase chemistry. The N-terminus of the fluorescent peptide was functionalized with a 2-nitrobenzyl alcohol moiety to introduce the photoresponsive trigger (Figure 3b, see SI for synthetic details). Next, a 15 wt % hydrazone hydrogel was formed by exposure of a solution of 10 mM of 1 and 5 mM of 2 to 365 light at 20 mW/cm2. Here, a stoichiometric excess of PEG-hydrazine 1 was utilized to ensure the presence of unreacted hydrazines for subsequent biochemical photopatterning. The resultant hydrogel was then swollen in a solution containing NB-K(fluorescein)GRGDS (10 mM) and then exposed to 365 nm light at 20 mW/cm2 for 5 min through a chrome photomask of 200 μm spaced stripes. After removal of the soluble/ unconjugated peptide, confocal microscopy images revealed the introduction of the RGDS ligand into 200 μm-striped green patterns (Figure 3c), and the pattern was clearly transferred throughout the depth of the hydrogel (200 μm, Figure 3d). These results indicate successful and facile conjugation of the RGD peptide in spatially defined regions throughout the hydrogel microenvironment. Further sequential immobilization of biomolecules should be possible so long as the hydrazine functionalities have not been completely consumed. Finally, we tested the cytocompatibility of the hydrazone cross-linking chemistry and the resultant hydrogel scaffold for three-dimensional primary cell culture studies with human mesenchymal stem cells (hMSCs), a cell type chosen for their mechanically and chemically driven fate decisions.27 Specifically, the hydrazone cross-linking reaction was performed in the presence of hMSCs so that they were encapsulated in the
Figure 4. Representative images for the Live/Dead assay performed 24 and 48 h postencapsulation (scale bar: 100 μm, green cells = live, red cells = dead, n = 9 images from 3 gels). Cells were encapsulated at 5 × 106 cells/mL in a mixed solution of 5 wt % PEG hydrazine 1, 5 wt % PEG-Nitrobenzyl 2, and 1 mM NB-KGRGDS. Insets show magnified image (scale bar 40 μm) and cell morphology.
we observed a small decrease in cell viability between 24 and 48 h, no statistically significant difference (p = 0.27) in cell viability was observed between 48 and 120 h, demonstrating the cytocompatibility of the hydrazone chemistry and the hydrogel system over extended periods of time. An added benefit of this system is the strong UV absorbing characteristics of the NB and resulting NB-aldehyde that protects the encapsulated cells from 22
DOI: 10.1021/acsmacrolett.5b00682 ACS Macro Lett. 2016, 5, 19−23
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exposure to 365 nm light during gelation. The encapsulated cells displayed a rounded morphology over the course of 120 h, whereas the cells on the surface of the gel exhibited a spread morphology. This observation can be explained by the low rate constant for hydrolysis of the aromatic hydrazone cross-link,16 which restricts cellular mobility in the scaffold. A more reversible, photogenerated aliphatic hydrazone cross-link could allow for cell spreading. Furthermore, the on-demand generation of aldehyde functionalities in this system could be used for in situ scaffold stiffening and patterning of photocaged biomolecules to exploit the biochemically and biophysically sensitive fate decisions of hMSCs, respectively, to control their differentiation with spatiotemporal control. In conclusion, we have demonstrated the synthesis of a new hydrazone bond forming photochemistry to generate cytocompatible hydrogel matrices for 3D cell encapsulation and spatial incorporation of biochemical moieties. Specifically, 2-nitrobenzyl alcohol based molecular components were utilized as photoresponsive moieties to form aldehydes in situ, which were then captured by hydrazine molecules to form hydrazone bonds. Results show that this photodriven hydrazone chemistry can be used to externally control not only the gel formation kinetics but also the final modulus of the hydrogel scaffold by simple variations in the light intensity and exposure time. Further, the effect of pH conditions indicated rapid hydrogel formation at physiological pH, suggesting the potential of this gel formation chemistry for mild and convenient cellular encapsulations. We then demonstrated the ability of the photochemistry to spatially pattern biochemical peptides within the hydrazone-based hydrogel system. Overall, a key advantage of the proposed hydrogel system is that it should enable researchers not only to fine-tune the mechanical properties of cell culture matrices by modulation of light intensity and exposure time but also to spatially modify the biological functionalities. This on-demand control should prove useful for probing cell responses to dynamic changes in matrix properties.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.5b00682. Additional synthetic details for polymeric precursors and experimental details for gelation, photopatterning, and cellular encapsulation studies (PDF)
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Letter
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions §
These authors contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Science Foundation (Grant NSF-DMR 1408955) and the Howard Hughes Medical Institute. 23
DOI: 10.1021/acsmacrolett.5b00682 ACS Macro Lett. 2016, 5, 19−23
