Mild chemoselective reactions with biomacromolecules are of significant interest and of widespread use in chemistry, biology, and more recently in nano-biotechnology. Such reactions can afford bioconjugates for characterization of native functions (e.g., electron-transfer proteins) (1), attachment to solid substrates (e.g., chemiluminescence biosensor applications) (2), preparation of biohybrid structures (e.g., derivatized bacteriophage) (3), and introduction of new activity (e.g., photoresponsive membrane channels) (4). Of these reactions, chemical ligation is a particularly attractive approach to prepare large proteins that are otherwise unattainable using conventional solid-phase peptide synthesis (5-8). Moreover this mild coupling reaction can be performed in the presence of functional groups in aqueous solutions. Recently, we used multiple thiazolidines as the chemical ligation between macromolecules to render a cross-linked hydrogel for the repair of acute corneal wounds (9). For applications where a hydrogel adhesive is required for longer periods, such as in a corneal transplant, we are evaluating the use of pseudoproline ligations between a dendron and poly(ethyleneglycol) possessing N-terminal cysteines and aldehyde esters, respectively. Herein we report the synthesis, characterization, and capability of this in situ polymerizing hydrogel to fasten corneal transplants in vitro.

Pseudoproline formation is one type of chemoselective and orthogonal peptide ligation method that has been applied successfully to the synthesis of a variety of proteins (5, 6, 10, 11). In this mild coupling strategy, a peptide possessing a C-terminal glycol aldehyde ester is reacted with another peptide possessing a N-terminal cysteine. The reaction occurs in a stepwise process with imine ligation and thiazolidine formation followed by an O,N-acyl migration to afford the pseudoproline linkage (Figure 1). For this approach to translate to a cross-linked network, two macromers are required where at least one possesses two and the other three or more reactive groups. Specifically, the dendron macromer (Figure 1) was prepared via successive amidations using the pentafluorophenyl activated esters of Lys (ZLys(Z)OPFP) and Cys (IsoCys(Boc)OPFP) (9). This macromer was selected because dendritic macromolecules provide a highly branched structure of defined composition with extensive surface functionalization capabilities (12-17), which facilitates subsequent intermolecular cross-linking to form a hydrogel. The two ester-aldehyde functionalities on the PEG macromer (MW 3400) were introduced as follows. The PEG diol was first reacted with succinic anhydride in pyridine to afford poly(ethylene glycol) disuccinic acid. Next, the cesium salt was prepared by neutralization of the acid with 1 M CsCO₃ until pH 8 followed by coupling bromoacetaldehyde dimethyl acetal in DMF to give the protected aldehyde. The acetal was deprotected using 10% TFA in DCM to afford the poly(ethylene glycol) diester-aldehyde (see Supporting Information for details).

Figure 1 (top) Reaction scheme for pseudoproline ligation to form cross-linked hydrogel. (bottom) poly(ethylene glycol) diester-aldehyde and N-terminal cysteine lysine dendron macromers.

Upon mixing an aqueous solution of the poly(ethylene glycol) diester-aldehyde and dendron macromers, a hydrogel (18, 19) forms within about 30 s (20, 33, or 50 wt % polymer solutions). Cylindrical hydrogel samples of 8 mm diameter and 3 mm thickness were prepared in aqueous buffer pH = 7.4, and the mechanical properties were measured at a frequency of 1 Hz. The hydrogels exhibited viscoelastic properties. The compressive (E) and complex modulus (G*) for the 20, 33, and 50 wt % hydrogels prepared were measured after 24 h of formation or after incubating for 24 h in an aqueous buffer solution at pH = 7.4 (Figure 2). As expected the mechanical properties increased with increasing macromer wt %. After swelling over 24 h, the moduli decrease relative to the unswollen hydrogels consistent with a more hydrated structure. The hydrogels are optically transparent with a UV cutoff of 300 nm (see Figure 4A).

	Figure 2 Compressive (E) and complex (G*) moduli for the various wt % hydrogels before and after swelling (sw) in neutral aqueous solution.
	Figure 3 Hydrogel weight loss as a function of time at 25 C when stored in a humidity chamber.
	Figure 4 (A) Photograph of the hydrogel. (B) Photograph of a corneal autograft fastened with 8 interrupted 10-0 nylon sutures. (C) photograph of a corneal autograft fastened with 8 interrupted 10-0 nylon sutures and the hydrogel sealant. (D) photograph after application of indy ink applied to a corneal autograft shown in C.

The hydrogel formed between the dendron and poly(ethylene glycol) diester-aldehyde is stable and retains its shape and size over time. As shown in Figure 3, this pseudoproline-cross-linked hydrogel remains intact for more than 6 months with less than a 10% change in weight when placed in a humidity chamber. At six months the value for compressive modulus is 90% of the original value, indicating that an extensive network is still present. This result is in contrast to a hydrogel formed between the dendron and poly(ethylene glycol) dialdehyde (9). In this later reaction, a hydrogel is formed via formation of multiple thiazolidines. This reaction represents the first half of the reaction sequence shown in Figure 1. Given that thiazolidine formation is reversible, the hydrogel is intact for relatively short periods. When placed in a humidity chamber, the thiazolidine-cross-linked hydrogel loses its original cylindrical shape and is an unstructured gelatinous mass at approximately one week (see Figure 3). Confirmation of the thiazolidine and the pseudoproline ligations was obtained using a model small molecular weight system in a manner analogous to that previously reported (see SI) (20).

We are interested in the design requirements and properties of hydrogels as sealants for the repair of ophthalmic wounds created as a result of injury, infection, or surgical procedure (21-25). For relatively small, acute wounds such as a cataract incision, the hydrogel adhesive formed via multiple thiazolidines (9) offers the advantages of securing the wound but not residing long-term at the wound site. In fact corneal re-epithelization of a small wound in humans occurs within about 3 days (26). However, for the repair of large corneal wounds such as corneal transplants, a sealant is desired that can remain longer at the wound site.

Corneal transplantation or penetrating keratoplasty (PKP) is one of the most common and successful tissue transplants (27). In a corneal transplantation the recipient cornea undergoes a large circular full-thickness cutting, or trephination, to remove the diseased or damaged tissue and then a previously trephinated donor corneal button is manually sutured to the recipient corneal rim. The major disadvantages related to this procedure include delayed visual recovery, suprachoroidal hemorrhage, neovascularization, microbial keratitis, postoperative suture removal (typically nine-months after transplantation), and surgically induced astigmatism (28-31). Furthermore, these complications in PKP are often a result of the use of sutures. A sutureless corneal transplant procedure would significantly improve PKP with regard to the multiple intraoperative and postoperative issues mentioned above. The idea of a sutureless keratoplasty is not new, but the development of a practical sutureless ophthalmic procedure has eluded the field since its original inception by Gradle in 1921, although there have been some encouraging results (32-34).

Toward this goal, we determined whether this in situ polymerizing hydrogel would reduce the number of sutures necessary to secure the incision between the host and graft corneal tissue. In this in vitro model, an 8 mm central corneal trephination was made in an enucleated eye and then this newly formed button was autografted back to the original eye. The host-graft tissue interface was secured using sutures, sutures plus the hydrogel sealant, or the hydrogel sealant alone (Figure 4). The leaking pressure for the autografted eyes was measured as we have done for corneal laceration studies to determine the extent to which the wound was sealed (9). The leaking pressure for autografts receiving 16 interrupted 10-0 nylon sutures was 13 ± 5 mmHg (n = 4). Today, this is a standard procedure for a PKP. Normal intraocular pressure (IOP) is 15 mmHg. When the hydrogel sealant was applied (33 wt %; 60 L) to the sutured wound with 16 interrupted sutures, the leaking pressure increased to 63 ± 7 mmHg (n = 4). Increasing the macromer wt % to 50% (60 L) with 16 interrupted sutures afforded a leaking pressure of 101 ± 5 mmHg (n = 4). Next, we evaluated if the hydrogel sealant in concert with 8 interrupted 10-0 nylon sutures would secure the wound. The autograft with 8 sutures had a leaking pressure of 5 mmHg or less (n = 4). When the hydrogel sealant at 33wt % % (60 L) and 8 sutures were used to secure the autograft, the wound leaked at a pressure of 45 ± 6 mmHg (n = 4). This leaking pressure approaches the upper bounds of the physiologically relevant limit, and thus we decided to increase the wt % of the polymers to attain a stronger seal. Application of the 50 wt % formulation (60 L) and 8 sutures afforded an autograft that leaked at a pressure of 77 ± 5 mmHg (n = 4). Application of the hydrogel sealant reduced the number of sutures required to secure the autograft. We were unable to secure the autograft to a safe high pressure when the hydrogel sealant was used alone, indicating that this hydrogel does not possess sufficient adhesivity by itself to secure a PKP. However, an additional benefit to this hydrogel sealant, beyond closing the wound with a reduced number of sutures, is the potential of the hydrogel barrier formed at the wound interface to protect the wound from postoperative infections. As a first indicator for the potential effectiveness of this hydrogel barrier, the transport of India ink across the hydrogel can be monitored as described by McDonnell (35). As shown in Figure 4D when India ink is applied to the wound, the dye does not penetrate into the anterior chamber, indicating that the wound interface is secured.

In summary, an in situ polymerizing hydrogel is described that forms in aqueous solution under mild conditions. This chemical-ligation strategy is attractive for hydrogel preparation since it allows synthetic flexibility in the use of a wide-range of macromer compositions, including potentially those with reactive groups, in a manner analogous to the reaction diversity obtained with previous bioconjugated proteins and enzymes. The hydrogel can be used in conjunction with a reduced number of sutures to successfully fasten a corneal transplant and secure the wound interface. The dendron macromer used in hydrogel formation belongs to a family of dendritic macromolecules composed of biocompatible building blocks (21, 22). This macromer system enables efficient cross-linking, varied hydrogel properties, and aqueous polymer solutions for application to a wound site. Dendritic macromolecules, due to their unique properties, structure, and high degree of compositional preciseness, are finding ever-increasing uses in medicine, ranging from vehicles for drug delivery and imaging agents to biomaterials for wound management and tissue restoration.

Acknowledgment

This work was supported by the NIH.