Michael A. Carnahan

In Situ Polymerized Hydrogels for Repairing Scleral Incisions Used in Pars Plana Vitrectomy Procedures

Crosslinked polymer networks possessing a high water content, otherwise known as hydrogels, are multipurpose materials for medical applications in areas such as drug delivery, tissue engineering, and wound healing. To form such hydrogels, dendritic or highly branched macromers are advantageous. These macromers provide opportunities to create hydrogels at low polymer concentration, to control swelling, and to vary the hydrogel mechanical properties. These favorACHTUNGTRENNUNGable attributes arise with dendritic polymers because of the well-defined composition, the large number of endgroups, the physiochemical properties (such as low viscosity), and the preparation methods that allow for precise structural control and optimization capabilities. We are synthesizing dendritic macromolecules composed of biocompatible building blocks and evaluating the corresponding hydrogels as ocular adhesives. Using a photocurable hydrogel system based on poly(glycerol-succinic acid)-polyethylene glycol hybrid dendritic-linear macromolecules, full-thickness 4.1 mm corneal lacerations in enucleated eyes and chicken eyes in vivo, as well as secured LASIK flaps in vitro, have been successfully repaired. In addition to light-activated hydrogel formation, other crosslinking strategies that quickly afford a hydrogel adhesive are also explored. Herein, we report the synthesis of lysineterm ACHTUNGTRENNUNGinated peptide dendrimers and dendrons, the formation of crosslinked hydrogels with a poly(ethylene glycol) di-activated ester, the analysis of hydrogel mechanical properties, and the closure of a sclerotomy incision—the wound created during a typical vitrectomy procedure. The dendrons ([G1]-Lys-NH2 and [G2]-Lys-NH2) and dendriACHTUNGTRENNUNGmers (([G1]-Lys-NH2)2-PEG and ([G2]-Lys-NH2)2-PEG) used for hydrogel formation were synthesized as shown in Scheme 1. Several amide-coupling approaches were explored (BOP, DCC, EDC, and oxalyl chloride), and the pentafluorophenol-ester strategy was found to yield the highest amide coupling reactions. Thus, we first prepared the pentafluorophenol-ester of ZLys(Z)OH and BocLysACHTUNGTRENNUNG(Boc)OH using N-N’-dicyclohexylcarbodiACHTUNGTRENNUNGimide (DCC) and 2,3,4,5,6-pentafluorophenol (PFP) in CH2Cl2. After crystallization in CH2Cl2/hexane, white crystalline products were obtained (98% and 92% yield, respectively). Next, ZLys(Z)OPFP was coupled to LysOMe·2HCl in the presence of diisopropylethylamine (DIEA) and 1-hydroxybenzotriazole (HOBT) to prevent racemization, to give 1. Compound 1 was purified by precipitation in ether and obtained in 98% yield. The Z-amino protecting groups were cleaved by hydrogenolysis (Pd/C, H2) in methanol (99% yield) and the amine functionality was subsequently acidified using 1m HCl (99% yield) to afford the [G1]-Lys-NH2 dendron, 2. The larger dendron [G2]-Lys-NH2, 3, was synthesized by reacting 2 with BocLysACHTUNGTRENNUNG(Boc)PFP in the presence of HOBT and DIEA, followed by treatment with TFA to remove the Boc protecting groups (70% and 99% yield, respectively). The protected intermediate was purified by silica gel chromatography (CH2Cl2/MeOH=95:5). The corresponding product obtained using ZLys(Z)OPFP was difficult to isolate and purify. The intermediates prepared in the dendron synthesis were also used to prepare the ([G1]-Lys-NH2)2-PEG, 4 and ([G2]-Lys-NH2)2-PEG 5 dendrimers. ZLys(Z)OPFP was coupled to diamino polyethylene glycol [MW=3400] in the presence of DIEA and HOBT in CH2Cl2, to afford ([G1]-Lys-NH2)2-PEG 4. The product was washed with water and purified by precipitation in ether (95% yield). The deprotection of the Z groups was achieved by hydrogenolysis (Pd/C, H2) in methanol followed by precipitation in ether to afford ([G1]-Lys-NH2)2-PEG 4 as a white powder (99% yield). The previously prepared, protected dendron 1 was used for the synthesis of the ([G2]-Lys-NH2)2-PEG dendrimer, 5. The methyl ester of 1 was hydrolyzed by saponACHTUNGTRENNUNGification with 1m NaOH in methanol followed by neutralization with 1m HCl (85% yield). The pentafluorophenol-ester of this acid was obtained by treatment with 2,3,4,5,6-pentafluorophenol and DCC in CH2Cl2 to afford a white powder after crystallization (95% yield). This activated ester dendron was then coupled to diamino polyethylene glycol [MW=3400] in CH2Cl2 to give, after precipitation in ether, a white powder in 96% yield. The Z groups were removed by hydrogenolysis and the product, ([G2]-Lys-NH2)2-PEG dendron 5, was isolated by precipitation in ether (99% yield). The structural identities of the various intermediates and dendritic macromolecules were determined by H, C NMR, and mass spectrometry (see Supporting Information). A hydrogel starts to form within one minute of mixing aqueous solutions of the dendrimer or dendron with a poly(ethylene glycol) disuccinimidyl proprionate, 6 ; PEG-NHS. The hydrogels are optically transparent and Figure 1 shows the 5 :6 hydrogel. All the hydrogels are formed using a 1:1 reactive group stoichiometry. The crosslinking reaction can be followed by infrared spectroscopy (IR), as shown in Figure 2a. For example, the IR stretch for the PEG-NHS at 1733 cm 1 disappears over time and the reaction between dendron 2 and 6 (1:1; 18 w/w%) is complete within 5000 seconds. As expected, the hydrogels swell when placed in an aqueous solution. Hydrogel swelling is linear with increasing concentrations of polymer [a] Dr. M. Wathier, Prof. M. W. Grinstaff Departments of Biomedical Engineering and Chemistry, Metcalf Center for Science and Engineering, Boston University, Boston, MA 02215 (USA) Fax: (+1)617-358-3186 E-mail : mgrin@bu.edu [b] Dr. M. S. Johnson, Dr. M. A. Carnahan, Dr. C. Baer, Dr. B. W. McCuen, Dr. T. Kim Department of Ophthalmology, Duke University Medical Center Durham NC 27710 (USA) [**] This work was supported by the NIH NEI. Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author.

Novel tissue adhesives to secure laser in situ keratomileusis flaps

Purpose: To evaluate 2 novel biodendrimer tissue adhesives in sealing and securing laser in situ keratomileusis (LASIK) flaps. Setting: Duke University Eye Center, Durham, North Carolina, USA. Methods: Laser in situ keratomileusis flaps were created in 10 human eye‐bank eyes using the Hansatome microkeratome system (Bausch & Lomb). These eyes were divided into 2 groups. Flaps in the first group (n = 4) were secured with a laser‐activated biodendrimer adhesive along the flap edge. In the second group (n = 6), the flaps were secured with a self‐gelling dendritic adhesive. Dry Merocel sponges (Medtronic Solan) were used to test the strength of flap adherence in both groups. Further testing was performed in the second group. The hinges of these flaps were cut with a scalpel blade and fluorescein dye was injected under the flap to observe potential dye leakage along the flap edge. Results: Laser in situ keratomileusis flaps sealed with both adhesives were secure with no flap dislocation. There was no leakage of fluorescein dye observed in the second group. Both adhesives were easy to apply, clear when dry, and had a soft rubbery consistency. Conclusions: Two novel biodendrimer adhesives successfully sealed and secured LASIK flaps. These adhesives may prove to be an effective alternative for treating LASIK flap complications such as epithelial ingrowth or flap dislocation.

Dendritic supramolecular assemblies for drug delivery

Dendritic supramolecular assemblies were formed in water with Reichardts dye or the anticancer drug 10-hydroxycamptothecin and the dendritic macromolecule, ([G4]-PGLSA-OH)2-PEG3400.