Michael R. Carrasco

Glucose-Permeable Interpenetrating Polymer Network Hydrogels for Corneal Implant Applications: A Pilot Study

Epithelialization of a keratoprosthesis requires that the implant material be sufficiently permeable to glucose. We have developed a poly(ethylene glycol)/poly(acrylic acid) (PEG/PAA) interpenetrating polymer network (IPN) hydrogel that can provide adequate passage of glucose from the aqueous humor to the epithelium in vivo. A series of PEG/PAA IPNs with varying PEG macromonomer molecular weights were synthesized and evaluated through swelling studies to determine their water content and diffusion experiments to assess their permeability to glucose. One of the PEG/PAA hydrogels prepared in this study had a glucose diffusion coefficient nearly identical to that of the human cornea (∼ 2.5 × 10− 6 cm2/sec). When implanted intrastromally in rabbit corneas, this hydrogel was retained and well-tolerated in 9 out of 10 cases for a period of 14 days. The retained hydrogels stayed optically clear and the epithelium remained intact and multilayered, indicating that the material facilitated glucose transport from the aqueous humor to the anterior part of the eye. The results from these experiments indicate that PEG/PAA hydrogels are promising candidates for corneal implant applications such as keratoprostheses and intracorneal lenses, and that the PEG/PAA IPN system in general is useful for creating permeable substrates for ophthalmic and other biomedical applications.

Synthesis of neoglycopeptides by chemoselective reaction of carbohydrates with peptides containing a novel N′-methyl-aminooxy amino acid

Abstract A novel N′-methyl-aminooxy amino acid has been designed, synthesized, and successfully incorporated into peptides using standard solid-phase peptide synthesis procedures. Reaction of these peptides with native reducing sugars yields neoglycopeptides via a chemoselective reaction with the aminooxy side chains. The key feature of the new amino acid is that it maintains attached sugars in their cyclic conformations and close to the peptide backbone.

Synthesis of Aminooxy and N-Alkylaminooxy Amines for Use in Bioconjugation

Five Boc-protected aminooxy and N-alkylaminooxy amines have been synthesized in 60-95% overall yield using a common synthetic strategy from readily available two- and three-carbon Cbz-protected amino alcohols. The amines can be linked to biomolecules via amide formation and incorporated directly into peptoids via submonomer synthesis. Subsequent deprotection of the aminooxy and N-alkylaminooxy groups enables conjugation with desired target molecules via established chemoselective ligation methods. The range of derivatives synthesized allows different distances to be established between the conjugated molecules.

Dap-SL: a new site-directed nitroxide spin labeling approach for determining structure and motions in synthesized peptides and proteins

A new approach for site‐directed placement of nitroxide spin labels in chemically synthesized peptides and proteins is described. The scheme takes advantage of a novel diaminopropionic acid scaffold to independently control backbone and side chain elongation. The result is a spin‐labeled side chain, referred to as Dap‐SL, in which an amide bond forms a linker between the nitroxide and the peptide backbone. The method was demonstrated in a series of helical peptides. Circular dichroism and nuclear magnetic resonance showed that Dap‐SL introduces only a minor perturbation in the helical structure. The electron paramagnetic resonance spectrum of the singly labeled species allowed for determination of the spin label rotational correlation time and suggests that the Dap‐SL side chain is more flexible than the modified Cys side chain frequently used in site‐directed spin label studies. Spectra of the doubly labeled peptides indicate a mixture of 310‐helix and α‐helix, which parallels findings from previous studies. The scheme demonstrated here offers a fundamentally new approach for introducing spin labels into proteins and promises to significantly extend biophysical investigations of large proteins and receptors. In addition, the technique is readily modified for incorporation of any biophysical probe.

Histological Processing of pH-Sensitive Hydrogels Used in Corneal Implant Applications

Abstract Hydrogels are hydrophilic, crosslinked polymers that are being used with increasing frequency in a variety of biomedical applications. The ability to obtain high-quality histological sections, without artifacts, after in vivo implantation is critical to their evaluation. Unfortunately, because of their high water content and environmental sensitivities, many hydrogels are unable to be successfully processed for histology using typical tissue processing methods. In this work, we present a method for successfully processing pHsensitive hydrogels for histological analysis. A modified glycol methacrylate (GMA) processing and embedding protocol is described. One cornea each from 10 New Zealand Red rabbits was implanted with a poly(ethylene glycol) and poly(acrylic acid) interpenetrating double network hydrogel disc for 2 weeks and processed for histology. Maintaining a neutral pH during fixation with glutaraldehyde is a critical initial step for processing. In addition, typical tissue processing methods require the use of alcohol and/or xylene for specimen dehydration, causing severe distortion of hydrogel morphology. The substitution of these chemicals with deionized water during the processing steps also proved to be vital for maintaining hydrogel structure and morphology. Slides stained with cresyl violet showed excellent tissue and hydrogel morphology with minimal histological artifacts or distortion (The J Histotechnol 30:157, 2007) Submitted August 27, 2006; accepted with revisions March 12, 2007.