Versatile click alginate hydrogels crosslinked via tetrazine–norbornene chemistry
Introduction
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 [1], [2], [3], [4], [5], [6], [7]. Alginate biopolymers are versatile, naturally derived linear polysaccharides comprised of repeating (1,4)-linked β-D-mannuronic and α-l-guluronic acid, and can be crosslinked to form hydrogels via a variety of ionic and covalent crosslinking methods [8], [9]. Alginate hydrogels can be engineered to release small molecules and proteins, present bioactive ligands to cells, and degrade at a tunable rate [10], [11], [12]. 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 [13].
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 Ca2+, to form ionic bridges between adjacent polymer chains. These gels have been shown to be weak and to lose mechanical 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 [14]. Calcium crosslinked alginate gels can yield non-uniform physical properties, due to extremely rapid crosslinking with certain ions [15]. Moreover, leached calcium from calcium crosslinked alginate gels can be immunostimulatory, which is unfavorable in many in vivo applications [16]. 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 [17].
To overcome many of the challenges associated with ionic crosslinking, alternative covalent crosslinking strategies have been developed, though none are completely biologically inert [18], [19], [20], [21]. 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 [22].
Click chemistry has recently emerged as an alternative approach to synthesize covalently crosslinked hydrogels with high chemoselectivity and fast reaction rates in complex aqueous media, at physiologically relevant pH and temperature ranges both in vitro and in vivo [23]. Recent findings have established a set of bioorthogonal 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 Diels–Alder reaction between tetrazine 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 [26], [27], [28], [29]. 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 crosslinked 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 covalent crosslinking without the need for external input of energy, crosslinkers, or catalysts, using the bioorthogonal inverse electron demand Diels–Alder 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 inflammatory response to click alginate hydrogels that are injected in vivo.
Section snippets
3-(p-benzylamino)-1,2,4,5 tetrazine synthesis
3-(p-benzylamino)-1,2,4,5-tetrazine was synthesized according to an established protocol [32]. 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
Synthesis, characterization, and crosslinking of click alginate polymers
To prepare click alginate polymers, norbornene or tetrazine groups were introduced to high molecular weight alginate biopolymers 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
Discussion
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 energy, crosslinkers, or catalysts. While recent work has used similar click chemistry for localized drug delivery, this work presents the first use of the tetrazine–norbornene click reaction to covalently crosslink polysaccharides into hydrogels [29], [38]. Crosslinking of alginate by different methods has
Conclusions
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 reaction 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
Acknowledgments
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 authors 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
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These authors contributed equally to this work.