Versatile click alginate hydrogels crosslinked via tetrazineenorbornene chemistry
Rajiv M. Desai a, b, 1, Sandeep T. Koshy a, b, c, 1, Scott A. Hilderbrand d, e, David J. Mooney a, b, *, Neel S. Joshi a, b, *
a School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA
b Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA c Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA
d Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114, USA
e Harvard Medical School, Boston, MA 02114, USA
Biomaterials 50 (2015) 30e37
Contents lists available at ScienceDirect Biomaterials
journal homepage: http://www.elsevier.com/locate/biomaterials
Received 3 November 2014 Accepted 20 January 2015 Available online
Click chemistry Cell adhesion
Cell encapsulation Tissue engineering
Hydrogels are highly hydrated, crosslinked polymer networks that resemble the environment of natural soft tissue, making them attractive materials for a variety of biomedical applications such as tissue engineering, drug delivery, and vaccines [1e7]. Alginate biopolymers are versatile, naturally derived linear polysaccharides comprised of repeating (1,4)-linked b-D-mannuronic and a-L- guluronic acid, and can be crosslinked to form hydrogels via a va- riety of ionic and covalent crosslinking methods [8,9]. Alginate hydrogels can be engineered to release small molecules and
* Corresponding authors. School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.
E-mail addresses: email@example.com (D.J. Mooney), njoshi@seas. harvard.edu (N.S. Joshi).
1 These authors contributed equally to this work.
0142-9612/© 2015 Elsevier Ltd. All rights reserved.
Alginate hydrogels are well-characterized, biologically inert materials that are used in many biomedical applications for the delivery of drugs, proteins, and cells. Unfortunately, canonical covalently crosslinked alginate hydrogels are formed using chemical strategies that can be biologically harmful due to their lack of chemoselectivity. In this work we introduce tetrazine and norbornene groups to alginate polymer chains and subsequently form covalently crosslinked click alginate hydrogels capable of encapsulating cells without damaging them. The rapid, bioorthogonal, and specific click reaction is irreversible and allows for easy incorporation of cells with high post-encapsulation viability. The swelling and mechanical properties of the click alginate hydrogel can be tuned via the total polymer concentration and the stoichiometric ratio of the complementary click functional groups. The click alginate hydrogel can be modified after gelation to display cell adhesion peptides for 2D cell culture using thiol-ene chemistry. Furthermore, click alginate hydrogels are minimally inflammatory, maintain structural integrity over several months, and reject cell infiltration when injected subcutaneously in mice. Click alginate hydro- gels combine the numerous benefits of alginate hydrogels with powerful bioorthogonal click chemistry for use in tissue engineering applications involving the stable encapsulation or delivery of cells or bioactive molecules.
© 2015 Elsevier Ltd. All rights reserved.
proteins, present bioactive ligands to cells, and degrade at a tunable rate [10e12]. Furthermore, ionically crosslinked alginates have been used extensively for drug delivery, cell encapsulation, and tissue engineering because ionic crosslinking can be largely benign to cells and encapsulated molecules .
The encapsulation of various small molecules, proteins, and cells in alginate hydrogels has thus far been largely limited to the reversible ionic crosslinking method which uses divalent cations, such as Ca2þ, to form ionic bridges between adjacent polymer chains. These gels have been shown to be weak and to lose me- chanical integrity over time in vitro and in vivo due to the reversible nature of the crosslinking and subsequent outward flux of ions from the hydrogel . Calcium crosslinked alginate gels can yield non- uniform physical properties, due to extremely rapid crosslinking with certain ions . Moreover, leached calcium from calcium crosslinked alginate gels can be immunostimulatory, which is un- favorable in many in vivo applications . While alginate is well
characterized in its ability to quantitatively couple small molecules, peptides, and proteins to the polymer backbone, these reactions (e.g. carbodiimide couplings) are typically limited in efficiency by slow reaction kinetics under aqueous conditions .
To overcome many of the challenges associated with ionic crosslinking, alternative covalent crosslinking strategies have been developed, though none are completely biologically inert [18e21]. Many of these covalent crosslinking strategies produce stable and uniform gels with mechanical properties that are controllable over a wider range compared to ionically crosslinked gels, but they may not be optimal for protein or cell encapsulation due to the cross- reactivity of the crosslinking chemistry with cells and proteins. Additionally, as the quantity and length of the crosslinker increases, the properties of the resulting hydrogel are significantly altered, making it difficult to compare such gels to alginate-based ionically crosslinked hydrogels .
Click chemistry has recently emerged as an alternative approach to synthesize covalently crosslinked hydrogels with high chemo- selectivity and fast reaction rates in complex aqueous media, at physiologically relevant pH and temperature ranges both in vitro and in vivo . Recent findings have established a set of bio- orthogonal click reactions that do not require the cytotoxic copper catalyst used in early reports. These copper-free chemistries include strain-promoted azide-alkyne cycloaddition (SPAAC) and the inverse electron demand DielseAlder reaction between tetra- zine and norbornene [24,25]. Previous reports have used these click reactions primarily to crosslink click end-functionalized branched polyethylene glycol (PEG) with linear crosslinkers composed of either PEG or linear peptides terminated with the appropriate click reaction pair [26e29]. The mechanical properties and swelling behavior of these click crosslinked PEG hydrogels could be tuned by varying the linear crosslinker concentration [30,31].
We hypothesized that a simpler and more robust click cross- linked biomaterial could be designed to exhibit stable and tunable mechanical properties, present bioactive ligands to cells, and encapsulate those cells in a cytocompatible covalent crosslinked alginate hydrogel. In this report, we modified alginate biopolymers with tetrazine or norbornene functional groups, allowing for co- valent crosslinking without the need for external input of energy, crosslinkers, or catalysts, using the bioorthogonal inverse electron demand DielseAlder click reaction. In addition to the crosslinking reaction, the click alginate system exploits photoinitated thiol-ene based modification of the norbornene groups to present thiol- bearing peptides. We investigated cell adhesion on the hydrogel surface and cell growth and viability when encapsulated in 3D in click alginate hydrogels. In addition, we studied the host inflam- matory response to click alginate hydrogels that are injected in vivo.
2. Materials and methods
2.1. 3-(p-benzylamino)-1,2,4,5 tetrazine synthesis
3-(p-benzylamino)-1,2,4,5-tetrazine was synthesized according to an estab- lished protocol . Briefly, 50 mmol of 4-(aminomethyl)benzonitrile hydrochloride and 150 mmol formamidine acetate were mixed while adding 1 mol of anhydrous hydrazine. The reaction was stirred at 80 C for 45 min and then cooled to room temperature, followed by addition of 0.5 mol of sodium nitrite in water. 10% HCl was then added dropwise to acidify the reaction to form the desired product. The oxidized acidic crude mixture was then extracted with DCM. After discarding the organic fractions, the aqueous layer was basified with NaHCO3, and immediately extracted again with DCM. The final product was then recovered by rotary evapo- ration, and purified by HPLC. All chemicals were purchased from Sigma-Aldrich.
2.2. Click alginate polymer synthesis
Click alginate biopolymers were modified with either 1-bicyclo[2.2.1]hept-5-en- 2-ylmethanamine (Norbornene Methanamine; Matrix Scientific) or 3-(p-benzyla- mino)-1,2,4,5-tetrazine by first allowing high molecular weight alginate, Mw 1⁄4 265 kDa (Protanol LF 20/40; FMC Technologies) to dissolve in stirred buffer containing 0.1 M MES, 0.3 M NaCl, pH 6.5 at 0.5% w/v. Next, N-hydroxysuccinimide
(NHS; Sigma-Aldrich) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hy- drochloride (EDC; Sigma-Aldrich) were added in 5 molar excess of the carboxylic acid groups of alginate. Either norbornene or tetrazine was then added at 1 mmol per gram of alginate to make Alg-N or Alg-T, respectively. The coupling reaction was stirred at room temperature for 24 h, after which the reaction was quenched with hydroxylamine (Sigma-Aldrich) and dialyzed in 12e14 kDa MWCO dialysis tubing (Spectrum Labs) for 4 days against a decreasing salt gradient from 150 mM to 0 mM NaCl in diH2O. The purified Alg-N and Alg-T polymers were treated with activated charcoal, sterile filtered (0.22 mm), and freeze-dried. This resulted in purified Alg-N or Alg-T polymers with a 5% degree of substitution of the available carboxylic acid groups of alginate (Fig. S-1).
2.3. Preparation and characterization of click alginate hydrogels
Click alginate hydrogels were prepared by first separately dissolving freeze- dried Alg-N and Alg-T polymers to final desired concentration (2e4% w/v) in Dul- becco’s Modified Eagle Medium (DMEM; Gibco). For gelation kinetics measure- ments, Alg-N and Alg-T polymer solutions were mixed at a desired ratio (i.e., 0.5e4:1 N:T) and directly pipetted onto the bottom plate of a TA Instruments ARG2 rheometer equipped with 8 mm flat upper plate geometry. A Peltier cooler was used to control the temperature for temperature dependent experiments, and mineral oil was applied to the gel periphery to prevent the hydrogel from drying during testing. Hydrogel samples were subjected to 1% strain at 1 Hz, and the storage and loss moduli (G0 and G00) were monitored for 4 h. For Young’s modulus measurements click alginate hydrogels were formed under siliconized glass plates (Sigmacote; Sigma-Aldrich) with 2 mm spacers. After 2 h of crosslinking at room temperature, cylindrical disks were punched using an 8 mm biopsy punch, transferred to DMEM, and swollen to equilibrium for 24 h at 37 C. Swollen hydrogel sample dimensions were measured using calipers for volumetric swelling ratio measurements, and then subjected to unconfined compression testing (1 mm/min) using a 10 N load cell with no preload (Instron Model 3342). The Young’s modulus, E, was calculated as the slope of the linear portion (first 10%) of the stress vs. strain curves.
2.4. Post-gelation thiol-ene photoreaction onto click alginate hydrogels
Click alginate hydrogels were made as previously described (2% w/v, N:T 1⁄4 2) and then a cell adhesive CGGGGRGDSP peptide (Peptide2.0) solution at 0.2 or 2 mM containing 0.5% w/v photoinitiator (Irgacure 2959; Sigma-Aldrich) was pipetted on top and the gel was covered with a glass coverslip. Gels were irradiated at 365 nm for 60 s at 10 mW/cm2. The gels were washed several times with DMEM to remove excess photoinitiator and unreacted peptide and swollen to equilibrium at 37 C before seeding with cells.
R.M. Desai et al. / Biomaterials 50 (2015) 30e37 31
EGFP 3T3 cell culture
NIH 3T3 (ATCC) cells were transduced with lentivirus produced from an EGFP- containing lentiviral vector (pLCAG EGFP, Inder Verma lab, Addgene plasmid 14857)  and were selected for 7 days in 1 mg/mL puromycin dihydrochloride (EMD Millipore). EGFP-expressing 3T3 fibroblast cells were cultured in DMEM supplemented with 10% (v/v) fetal calf serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco) at 37 C, in a 5% CO2 environment. Cells were passaged approximately twice per week.
For cell adhesion studies, slabs of click alginate hydrogels were modified with cell adhesion peptides as described above. 6 mm disks were punched, placed in DMEM, washed several times, and swollen for 4 h prior to seeding with cells at 5 104 cells/mL at a depth of approximately 1 mm above the surface of the gel. Cells were given 24 h to adhere and spread and then visualized via EGFP fluorescence using an epifluorescence microscope. EGFP images were used to quantify total cell area using ImageJ software. After 3 days of culture, cells were fixed and stained using Alexa Fluor 594 phalloidin (Molecular Probes) and Hoescht 33342 (Molecular Probes) to visualize F-actin filaments and nuclei respectively. To visualize cell death, gels were incubated for 20 min with a 4 mM ethidium homodimer-1 (Molecular Probes) solution in Hanks Buffered Saline Solution (HBSS) and imaged using an epifluorescence microscope.
For cell encapsulation studies, Alg-N polymers were modified to have approxi- mately 20 cell adhesive GGGGRGDSP peptides (Peptide2.0) per alginate chain as previously described . 600 mm thick click alginate hydrogels at 2% w/v, N:T 1⁄4 1, were then made containing cells at 3 106 cells/mL. Ionically crosslinked hydrogels were similarly prepared at 2% w/v using the same cell density and backbone RGD modified Alg-N polymers. A CaSO4 slurry (0.21 g CaSO4/mL ddH2O) at a final con- centration of 2% w/v was used to crosslink the ionically crosslinked hydrogel sam- ples so as to match the mechanical properties of the two substrates as closely as possible. To minimize the time in which cells did not have access to culture media, gels were allowed to crosslink at room temperature for 1 h, after which 6 mm disks
32 R.M. Desai et al. / Biomaterials 50 (2015) 30e37
were punched and placed in culture medium where the crosslinking reaction was expected to proceed to completion.
2.8. 3D in vitro cell assays
Cells were retrieved from alginate hydrogels by digestion in a 5 U/mL alginate lyase (Sigma-Aldrich) solution in HBSS for 20 min. For viability testing, cells were stained with a Muse Count and Viability Kit and tested on a Muse Cell Analyzer (EMD Millipore). To assess total cell metabolic activity, gels were transferred to wells containing 10% AlamarBlue (AbD Serotec) in cell culture medium and incubated for 4 h. The reduction of AlamarBlue was assessed according to the manufacturer’s instructions.
All work was done with BALB/cJ mice (female, aged 6e8 weeks; Jackson Labo- ratories) and was performed in compliance with National Institutes of Health and institutional guidelines.
2.10. In vivo hydrogel inflammatory response
Ultrapure alginate with low endotoxin levels (MVG alginate, ProNova Biomed- ical AS) was modified as described above with norbornene and tetrazine and sub- sequently prepared at 2% w/v in DMEM after purification. Click alginate hydrogels were prepared by mixing ultrapure Alg-N and Alg-T polymers with N:T 1⁄4 1 by connecting two syringes with a luer lock. 15 min after mixing, 50 uL of click alginate hydrogel was injected subcutaneously through an 18G needle. For ionic hydrogel samples, a 2% w/v ultrapure alginate solution was prepared in DMEM and similarly mixed in a syringe with a CaSO4 slurry at a final concentration of 2%. 50 uL of the ionically crosslinked gel was also injected subcutaneously in the same mice. Both gel samples were retrieved along with the surrounding skin after 1 week, 1 month, and 2 months of injection and fixed overnight in 10% neutral buffered formalin solution (Sigma-Aldrich). Samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) by the Harvard Rodent Histopathology Core.
3.1. Synthesis, characterization, and crosslinking of click alginate polymers
To prepare click alginate polymers, norbornene or tetrazine groups were introduced to high molecular weight alginate bio- polymers using conventional carbodiimide chemistry (Fig. 1-A). The degree of substitution of norbornene or tetrazine groups onto purified click alginate polymers was determined from 1H NMR spectra (Fig. S-1). A 5% degree of substitution of norbornene (Alg-N) or tetrazine (Alg-T) on alginate carboxyl groups was obtained using this method, and these batches of click alginate polymers were used for all subsequent experiments.
O O HO O OH O HO O O
O NH O NH
Alg-T N NNN
O OHOOHOOHO O
OH O HO O O OOH OOH
O OHOOHOOHO O
OH O HO O O OOH OOH
O NH O NH
To form click alginate hydrogels, Alg-N and Alg-T polymer so- Alg-N lutions were prepared separately and mixed together to gel. Upon
mixing of the two click alginate polymers, a stable gel was formed
via an inverse electron demand DielseAlder reaction between the
Alg-T N NN
two polymers, which releases nitrogen gas (Fig. 1-B). The nitrogen gas evolved from the crosslinking reaction does lead to the for- mation of a few small bubbles within the hydrogel. A stable gel was formed within 1 h at 25 C (Fig. 2-A), though the gelation kinetics could be tuned by varying the temperature or initial degree of substitution of the click alginate polymers (data not shown). The gelation kinetics at 25 C are favorable because it allows the user to easily achieve a well-mixed polymer formulation before gelation, a common challenge with other alginate hydrogel crosslinking methods.
3.2. Compressive Young’s modulus and swelling behavior
The mechanical properties of the extracellular matrix have been shown to affect cell fate and function in 2D and 3D environments [34e37]. In order to tune mechanical properties over a wide range, click alginate polymers were mixed at different ratios of Alg-N and Alg-T (N:T ratio) for a given polymer concentration between 2 and 4% w/v. These click alginate hydrogel samples were subjected to
Click Alginate Hydrogel
Fig. 1. Fabrication of click alginate hydrogels. Schematic of click alginate polymer synthesis. Aqueous carbodiimide chemistry is used to modify alginate backbone car- boxylic acids with tetrazine or norbornene, resulting in Alg-T or Alg-N polymers respectively (A). Alg-T and Alg-N polymers are mixed together to create a covalently crosslinked click alginate hydrogel network, with the loss of N2 (B).
A 1000 100 10
B, Table S-1, Table S-2). The ability to tune the mechanical proper- ties of the resulting gel over a large range by simply changing the ratio of the two polymers allows control over gel stiffness while keeping other parameters such as polymer concentration, and ligand density constant which may be useful for studies of mechanobiology.
The swelling ratio of hydrogel systems can affect mechanical properties, mass transport, and the presentation of ligands on the gel surface. To investigate how volumetric swelling would change at different polymer concentrations and N:T ratios, click alginate hydrogels were made as previously described and allowed to swell for 24 h at 37 C. The swollen volume was measured and compared to the casted volume (Fig. 2-C). For a given polymer concentration, the volumetric swelling ratio increased as the N:T ratio deviated from 1, demonstrating an inverse relationship between mechanical properties and swelling ratio as expected. While the N:T ratio has a significant effect on the swelling ratio, the polymer concentration does not have a significant effect, indicating that the swelling ratio of click alginate is dominated by crosslink density rather than polymer concentration (Table S-3).
3.3. Post-gelation modification of click alginate hydrogels
To explore if additional functionalities can be introduced to click alginate hydrogels after polymerization, we grafted thiol- containing molecules onto unreacted norbornenes in pre-formed click alginate hydrogels using a photoinitiated thiol-ene reaction (Fig. 3-A). Gels with N:T 1⁄4 2 were used to ensure unreacted nor- bornenes were available to react after the initial gelation. RGD peptide solutions at high (2 mM) or low (0.2 mM) concentration were reacted onto the surface of these click alginate hydrogels, which were then seeded with NIH 3T3 fibroblasts expressing a cytosolic fluorescent marker (EGFP). 3T3 cells readily adhered and spread on gels modified with RGD, while very few cells were able to attach or elongate on control gels with no RGD (Fig. 3-B). Cells on click alginate hydrogels presenting RGD were able to form branched interconnected networks, with a significant RGD density- dependent 2e3 fold increase in surface coverage over the 3 day culture, while unmodified click alginate gels were observed to be non-cell-adhesive and showed a decrease in surface coverage by cells over time (Fig. 3-C). After 3 days in culture, cells also showed an increase in spreading and actin stress fiber formation with higher RGD concentration (Fig. 3-D). Additionally, the high viability of cells after 3 days of culture demonstrated the cytocompatibility of the click alginate hydrogels for 2D cell culture (Fig. 3-E).
3.4. Cell encapsulation in click alginate hydrogels
In order to demonstrate the utility of click alginate hydrogels for cell encapsulation, cell viability and metabolic activity of cells encapsulated in click alginate hydrogels were investigated over a 3 day culture period; ionically crosslinked hydrogels were used for comparison in these studies. Representative images of encapsu- lated cells stained with ethidium homodimer-1 show minimal cell death in both click and ionically crosslinked gels 4 h and 3 days after encapsulation (Fig. 4-A). Quantification revealed that click alginate hydrogels resulted in significantly higher viability of encapsulated 3T3 cells both immediately after encapsulation (93±1%vs.87±2%)andafter3daysofculture(84±2%vs.79±4%) (Fig. 4-B). It should be noted that a loss in measured cell viability may occur during the cell retrieval process by enzymatic digestion of the hydrogels. The overall metabolic activity of the cells encap- sulated in the different hydrogels was also analyzed, and noted to increase over the 3 day culture period for both hydrogel cross- linking chemistries (Fig. 4-C).
B 100 10 1
Storage Modulus, G’ Loss Modulus, G”
10 100 200 300
0.10 2 4 6
Fig. 2. Click alginate hydrogel mechanical properties. Representative in situ dynamic rheometry plot at 25 C for 3% w/v click alginate at N:T 1⁄4 1, demonstrating modulus evolution with time (A). Compressive Young’s modulus (B) and volumetric swelling ratios (C) for 2%, 3% and 4% w/v click alginate hydrogels at varying N:T ratio. Values represent mean and standard deviation (n 1⁄4 4).
unconfined compression tests resulting in a compressive Young’s modulus that predictably increased with increasing polymer con- centration, and decreased as the ratio between the polymers deviated from the stoichiometrically balanced N:T ratio of 1 (Fig. 2-
R.M. Desai et al. / Biomaterials 50 (2015) 30e37 33
Young’s Modulus (kPa)
Shear Modulus (Pa)
R.M. Desai et al. / Biomaterials 50 (2015) 30e37
80 60 40 20
High RGD Low RGD No RGD
Photoinitiator + UV
HS R NH2
R = GGGGRGDSP
Fig. 3. Cell adhesion, spreading, and proliferation on click alginate hydrogels modified with RGD peptides after synthesis. Schematic of CGGGGRGDSP peptide coupling reaction onto click alginate hydrogel surface using photoinitiated thiol-ene chemistry (A). Representative images of 3T3 fibroblast adhesion, spreading, and proliferation on click alginate hydrogels with varying RGD peptide density (scale bar 1⁄4 200 mm) (B), and quantification (Two-Way ANOVA with Turkey’s post-hoc test, *p < 0.05, ****p < 0.0001 relative to No RGD control; Values represent mean and standard deviation, n 1⁄4 4e7) by endogenous EGFP expression (green) over 3 days (C). Phalloidin (red) and Hoescht 33342 (blue) staining of F- actin filaments and nuclei at 3 days for cells adherent to RGD modified click alginate hydrogels (scale bar 1⁄4 100 mm) (D). Representative fluorescent images of EGFP (green) 3T3 cells cultured on click alginate hydrogels with varying ligand density for 3 days and stained with ethidium homodimer-1 (red) (scale bar 1⁄4 100 mm) (E). The High, Low, and No RGD conditions refer to the 2 mM, 0.2 mM, and 0 mM peptide solutions used to modify the click alginate hydrogel surface. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.5. In vivo injection
The inflammatory response to the injection of click alginate hydrogels in vivo was investigated next. Click crosslinked and ion- ically crosslinked alginate hydrogels were injected subcutaneously and retrieved after 1 week, 1 month, and 2 months. The gelation kinetics of click alginate hydrogels allows them to be mixed and readily injected, in a similar manner to ionically crosslinked
hydrogels. A thin fibrous capsule was found to surround both types of gels 1 week after injection. H&E staining revealed a very thin capsule of collagen and fibroblasts surrounding the material throughout the duration of the study with minimal inflammation (Fig. 5). At 1 month, the ionically crosslinked gels were seen to lose structural integrity and allowed for infiltration of fibroblasts and immune cells into the gel, while the click crosslinked samples showed no evidence of breakdown nor cell infiltration into the
No RGD Low RGD High RGD
No RGD Low RGD High RGD
Surface Coverage (%)
R.M. Desai et al. / Biomaterials 50 (2015) 30e37 35
100 80 60 40 20 0
Fig. 4. Cell encapsulation in click crosslinked and ionically crosslinked alginate hydrogels. 3T3 fibroblasts were encapsulated in 2% w/v click crosslinked (N:T 1⁄4 1) and ionically crosslinked alginate hydrogels and stained with ethidium homodimer-1 (red) for dead cells at 4 h and 3 days post-encapsulation (scale bar 1⁄4 100 mm) (A). Quantitative analysis of cell viability (Two-Way ANOVA with Sidak’s post-hoc test, **p < 0.01, ***p < 0.001; Values represent mean and standard deviation, n 1⁄4 4) and overall metabolic activity as measured by reduction of AlamarBlue over time in culture (n 1⁄4 6) (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
material for up to 2 months (see Fig. S-2), and maintained a thin layer of fibroblasts surrounding the gel.
Our results show that alginate polymers can be modified with norbornene and tetrazine to create alginate hydrogels with a wide- range of mechanical properties without the input of external en- ergy, crosslinkers, or catalysts. While recent work has used similar click chemistry for localized drug delivery, this work presents the first use of the tetrazineenorbornene click reaction to covalently crosslink polysaccharides into hydrogels [29,38]. Crosslinking of alginate by different methods has been extensively explored to make covalently crosslinked hydrogels that are mechanically robust, but these chemistries lack the cytocompatibility inherent in
the bioorthogonal click reaction reported here [19,21,39]. The simplicity of this crosslinking modality provides the opportunity to control the mechanical properties of the click alginate hydrogel by adjusting the ratio of the polymers, rather than changing the total concentration of polymers in the system. This could potentially allow for the decoupling of material variables such as gel archi- tecture, stiffness, and ligand density in further applications of click alginate hydrogels.
Click crosslinked alginate hydrogels were used to form a cyto- compatible 2D cell culture substrate that can be modified to display cell adhesion peptides at varying concentrations. Alginate hydro- gels must display cell adhesive ligands in order for mammalian cells to attach, spread, and proliferate on the surface of the hydrogel. Without ligands such as RGD presented from the hydrogel surface, few cells will attach, and those that do will retain a spherical
Viable Cells (%)
AlamarBlue Reduction (%)
36 R.M. Desai et al. / Biomaterials 50 (2015) 30e37
Fig. 5. Tissue response following subcutaneous injection of click and ionically crosslinked hydrogels in vivo. Representative hematoxylin and eosin (H&E) stain of tissue sections at 1 week, 1 month, and 2 month following injection into BALB/cJ mice (scale bar 1⁄4 150 mm). Images focus on the geletissue interface, with dashed lines indicating the border between the hydrogel and the surrounding tissue. Asterisks indicate the location of the click alginate hydrogel, which separates from the tissue during histological analysis with no cell infiltration.
morphology and undergo apoptosis . Unfortunately, the car- bodiimide chemical reaction most commonly used to attach RGD peptides to the backbone of alginate is slow and requires lengthy purification and lyophillization time . In this work, photo- initated thiol-ene chemistry between norbornene and cysteine- bearing RGD peptides was employed to rapidly modify click algi- nate hydrogels to present adhesion ligands on the surface of the gel. This thiol-ene reaction is a powerful light-mediated click reaction that is simple, reproducible, fast, and highly efficient e achieving conversions nearing completion in aqueous media . Although we did not investigate the thiol-ene reaction conversion as a function of hydrogel depth specifically, several recent papers have reported the ability to functionalize the interiors of hydrogels using this method [28,30,42,43]. When click alginate hydrogels were modified with RGD peptides using this strategy, fibroblasts seeded on the gels responded with increased attachment and spreading as RGD density was raised, over a 3 day culture period. In addition to the simple and rapid coupling reaction, the thiol-ene based strategy for modifying alginate hydrogels also presents a straightforward method to change the ligand density on hydrogels of otherwise equal composition. Altogether, these data demonstrate the flexi- bility of click alginate hydrogels for culturing cells in 2D and allowing independent control over the presentation of bioactive ligands on the gel surface.
Furthermore, click crosslinked alginates can be used in vitro to encapsulate cells in 3D with high viability, providing a covalent alternative to conventional ionically crosslinked alginate hydrogels. A variety of cell types have been encapsulated in ionically cross- linked RGD modified alginates with high viability in vitro [11,35,44e46]. However, encapsulation of cells in covalently crosslinked RGD modified alginates is limited by the potential in- compatibility of the available crosslinking chemistries [47,48]. The data shown here establishes the ability to encapsulate fibroblasts in covalently crosslinked RGD modified click alginate hydrogels while maintaining cell viability at a high level. The aforementioned ability to independently tune the microenvironment mechanical proper- ties and adhesion ligand density can be exploited with the click crosslinked 3D cell culture system in the future to probe cell re- sponses to a variety of stimuli in vitro.
In vivo testing showed that click alginate hydrogels can crosslink in situ, provoke minimal inflammatory response, and resist frag- mentation and cell infiltration when injected subcutaneously. Histology revealed minimal acute inflammation in the tissue sur- rounding the injected gel in both click crosslinked and ionically crosslinked alginate. As is typical with many biomaterials, a small fibrotic capsule was formed around the hydrogel periphery in both cases . When compared to ionically crosslinked alginate, click alginate hydrogels demonstrate superior long-term structural integrity. Ionically crosslinked samples fragmented significantly after 1 month in vivo, resulting in cell infiltration, whereas the click alginate hydrogels remained intact during the 2 month study and were highly resistant to cell infiltration. In tissue engineering ap- plications where cell trafficking within the hydrogel is desirable, click alginate hydrogels could be processed using existing tech- niques to introduce microscale porosity to the hydrogels [50,51]. Alternatively, click alginate polymers could be crosslinked using tetrazine or norbornene-modified matrix metalloproteinase- degradable peptide sequences to allow cell-mediated degradation [29,52]. The use of partially oxidized alginate polymers would also allow degradation of the hydrogel over controlled time scales for in vivo tissue engineering applications [20,53]. The tissue compat- ibility and stability of click alginate hydrogels could make it particularly useful for applications where isolation from host im- mune cell infiltration is required [54,55].
Click alginate polymers are synthetically accessible and can be crosslinked in biological media at physiological pH to create tunable hydrogels with a wide range of mechanical properties. The rapid, bioorthogonal, and cytocompatible click crosslinking reac- tion makes click alginate hydrogels favorable for cell engineering applications. Click alginate hydrogels can be quickly modified to be cell adhesive and used for 2D or 3D cell culture. Additionally, click alginates have a minimal inflammatory response and high stability in vivo, making them attractive materials to use for long-term cell encapsulation and biomaterials-based tissue engineering applications.
This work was supported by the Army Research Office (W911NF- 13-1-0242) and the NIH (R01 DE013349). This work was performed in part at the MGH Center for Systems Biology. The authors would like to acknowledge the help of Olivier Kister, Kaixiang Lin, and Chris Johnson for material synthesis and troubleshooting. The au- thors would also like to thank Dr. Luo Gu, Dr. Ovijit Chaudhuri, Daniel Rubin, Alexander Cheung, Dr. Catia Verbeke, Zsofia Botiyanski, Ajay Parmar, and Max Darnell for scientific discussions.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.01.048.
-  Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920e6.
-  Ratner BD, Bryant SJ. Biomaterials: where we have been and where we are
going. Annu Rev Biomed Eng 2004;6:41e75.
-  Drury JL, Mooney DJ. Hydrogels for tissue engineering: scaffold design vari-
ables and applications. Biomaterials 2003;24:4337e51.
-  Kearney CJ, Mooney DJ. Macroscale delivery systems for molecular and
cellular payloads. Nat Mater 2013;12:1004e17.
-  Conway A, Schaffer DV. Biomaterial microenvironments to support the gen-
eration of new neurons in the adult brain. Stem Cells 2014;32:1220e9.
-  Huebsch N, Kearney CJ, Zhao X, Kim J, Cezar CA, Suo Z, et al. Ultrasound- triggered disruption and self-healing of reversibly cross-linked hydrogels for drug delivery and enhanced chemotherapy. Proc Natl Acad Sci 2014;111:
-  Hori Y, Winans AM, Huang CC, Horrigan EM, Irvine DJ. Injectable dendritic
cell-carrying alginate gels for immunization and immunotherapy. Bio-
-  Martinsen A, Skjåk-Braek G, Smidsrød O. Alginate as immobilization material:
I. Correlation between chemical and physical properties of alginate gel beads.
Biotechnol Bioeng 1989;33:79e89.
-  Augst AD, Kong HJ, Mooney DJ. Alginate hydrogels as biomaterials. Macromol
-  Freeman I, Kedem A, Cohen S. The effect of sulfation of alginate hydrogels on
the specific binding and controlled release of heparin-binding proteins. Bio-
-  Madl CM, Mehta M, Duda GN, Heilshorn SC, Mooney DJ. Presentation of BMP-
2 mimicking peptides in 3D hydrogels directs cell fate commitment in oste-
oblasts and mesenchymal stem cells. Biomacromolecules 2014;15:445e55.
-  Boontheekul T, Kong HJ, Mooney DJ. Controlling alginate gel degradation utilizing partial oxidation and bimodal molecular weight distribution. Bio-
-  Coviello T, Matricardi P, Marianecci C, Alhaique F. Polysaccharide hydrogels
for modified release formulations. J Control Release 2007;119:5e24.
-  Shoichet MS, Li RH, White ML, Winn SR. Stability of hydrogels used in cell encapsulation: an in vitro comparison of alginate and agarose. Biotechnol
-  Kuo CK, Ma PX. Ionically crosslinked alginate hydrogels as scaffolds for tissue
engineering: Part 1. Structure, gelation rate and mechanical properties. Bio-
-  Chan G, Mooney DJ. Ca(2+) released from calcium alginate gels can promote
inflammatory responses in vitro and in vivo. Acta Biomater 2013;9:9281e91.
-  Rowley JA, Madlambayan G, Mooney DJ. Alginate hydrogels as synthetic
extracellular matrix materials. Biomaterials 1999;20:45e53.
-  Seliktar D. Designing cell-compatible hydrogels for biomedical applications.
-  Eiselt P, Lee KY, Mooney DJ. Rigidity of two-component hydrogels prepared
from alginate and poly(ethylene glycol)%diamines. Macromolecules 1999;32:
-  Bouhadir KH, Hausman DS, Mooney DJ. Synthesis of cross-linked poly (alde-
hyde guluronate) hydrogels. Polymer 1999;40:3575e84.
-  Jeon O, Bouhadir KH, Mansour JM, Alsberg E. Photocrosslinked alginate hydrogels with tunable biodegradation rates and mechanical properties.
-  Lee KY, Rowley JA, Eiselt P, Moy EM, Bouhadir KH, Mooney DJ. Controlling
mechanical and swelling properties of alginate hydrogels independently by cross-linker type and cross-linking density. Macromolecules 2000;33: 4291e4.
-  Tibbitt MW, Anseth KS. Dynamic microenvironments: the fourth dimension. Sci Transl Med 2012;4. 160ps24e4.
-  Jewett JC, Bertozzi CR. Cu-free click cycloaddition reactions in chemical biology. Chem Soc Rev 2010;39:1272e9.
 Devaraj NK, Weissleder R, Hilderbrand SA. Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjugate Chem 2008;19:2297e9.  DeForest CA, Anseth KS. Cytocompatible click-based hydrogels with dynam- ically tunable properties through orthogonal photoconjugation and photo-
cleavage reactions. Nat Chem 2011;3:925e31.
 DeForest CA, Polizzotti BD, Anseth KS. Sequential click reactions for synthe-
sizing and patterning three-dimensional cell microenvironments. Nat Mater
 Fairbanks BD, Schwartz MP, Halevi AE, Nuttelman CR, Bowman CN, Anseth KS.
A versatile synthetic extracellular matrix mimic via thiol-norbornene photo-
polymerization. Adv Mater 2009;21:5005e10.
hydrogels for three-dimensional cell culture formed using tetrazi-
neenorbornene chemistry. Biomacromolecules 2013;14:949e53.
 Aimetti AA, Machen AJ, Anseth KS. Poly(ethylene glycol) hydrogels formed by thiol-ene photopolymerization for enzyme-responsive protein delivery. Bio-
by thiol-ene photoclick chemistry. Biomacromolecules 2012;13:2003e12.  Karver MR, Weissleder R, Hilderbrand SA. Synthesis and evaluation of a series of 1,2,4,5-tetrazines for bioorthogonal conjugation. Bioconjugate Chem
 Pfeifer A, Ikawa M, Dayn Y, Verma IM. Transgenesis by lentiviral vectors: lack
of gene silencing in mammalian embryonic stem cells and preimplantation
embryos. Proc Natl Acad Sci U S A 2002;99:2140e5.
 Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell
lineage specification. Cell 2006;126:677e89.
 Huebsch N, Arany PR, Mao AS, Shvartsman D, Ali OA, Bencherif SA, et al.
Harnessing traction-mediated manipulation of the cell/matrix interface to
control stem-cell fate. Nat Mater 2010;9:518e26.
 Khetan S, Guvendiren M, Legant WR, Cohen DM, Chen CS, Burdick JA.
Degradation-mediated cellular traction directsstem cell fate in covalently
crosslinkedthree-dimensional hydrogels. Nat Mater 2013;12:1e8.
 Chaudhuri O, Koshy ST, Branco da Cunha C, Shin J-W, Verbeke CS, Allison KH, et al. Extracellular matrix stiffness and composition jointly regulate the in- duction of malignant phenotypes in mammary epithelium. Nat Mater
 Mejía Oneto JM, Gupta M, Leach JK, Lee M, Sutcliffe JL. Implantable biomaterial
based on click chemistry for targeting small molecules. Acta Biomater
 Lee KY, Bouhadir KH, Mooney DJ. Controlled degradation of hydrogels using
multi-functional cross-linking molecules. Biomaterials 2004;25:2461e6.
 Rowley JA, Mooney DJ. Alginate type and RGD density control myoblast
phenotype. J Biomed Mater Res 2002;60:217e23.
 Hoyle CE, Bowman CN. Thiol-ene click chemistry. Angew Chem Int Ed
 Gramlich WM, Kim IL, Burdick JA. Synthesis and orthogonal photopatterning
of hyaluronic acid hydrogels with thiol-norbornene chemistry. Biomaterials
 Mu~noz Z, Shih H, Lin C-C. Gelatin hydrogels formed by orthogonal thio-
lenorbornene photochemistry for cell encapsulation. Biomater Sci 2014;2:
 Fonseca KB, Gomes DB, Lee K, Santos SG, Sousa A, Silva EA, et al. Injectable
MMP-sensitive alginate hydrogels as hMSC delivery systems. Bio-
 Nakaoka R, Hirano Y, Mooney DJ, Tsuchiya T, Matsuoka A. Study on the po-
tential of RGD- and PHSRN-modified alginates as artificial extracellular
matrices for engineering bone. J Artif Organs 2013;16:284e93.
 Kreeger PK, Deck JW, Woodruff TK, Shea LD. The in vitro regulation of ovarian follicle development using alginate-extracellular matrix gels. Biomaterials
 Lee KY, Alsberg E, Mooney DJ. Degradable and injectable poly(aldehyde
guluronate) hydrogels for bone tissue engineering. J Biomed Mater Res
 Jeon O, Alsberg E. Photofunctionalization of alginate hydrogels to promote
adhesion and proliferation of human mesenchymal stem cells. Tissue Eng Part
 Mikos A, McIntire L, Anderson J, Babensee J. Host response to tissue engi-
neered devices. Adv Drug Deliv Rev 1998;33:111e39.
 Annabi N, Nichol JW, Zhong X, Ji C, Koshy S, Khademhosseini A, et al. Con-
trolling the porosity and microarchitecture of hydrogels for tissue engineer-
ing. Tissue Eng Part B Rev 2010;16:371e83.
 Koshy ST, Ferrante TC, Lewin SA, Mooney DJ. Injectable, porous, and cell-
responsive gelatin cryogels. Biomaterials 2014;35:2477e87.
 Lutolf MP, Raeber GP, Zisch AH, Tirelli N, Hubbell JA. Cell-responsive synthetic
hydrogels. Adv Mater Weinh 2003;15:888e92.
 Lee KY, Bouhadir KH, Mooney DJ. Degradation behavior of covalently cross-linked
poly (aldehyde guluronate) hydrogels. Macromolecules 2000;33:97e101.
 Jacobs-Tulleneers-Thevissen D, Chintinne M, Ling Z, Gillard P, Schoonjans L, Delvaux G, et al. Sustained function of alginate-encapsulated human islet cell implants in the peritoneal cavity of mice leading to a pilot study in a type 1
diabetic patient. Diabetologia 2013;56:1605e14.
 Ma M, Chiu A, Sahay G, Doloff JC, Dholakia N, Thakrar R, et al. Core-shell
hydrogel microcapsules for improved islets encapsulation. Adv Healthc Mater 2013;2:667e72.
R.M. Desai et al. / Biomaterials 50 (2015) 30e37 37