The release kinetics for a variety of proteins of a wide range of molecular mass, hydrodynamic radii, and isoelectric points through a nanofiber hydrogel scaffold consisting of designer self-assembling peptides were studied by using single-molecule fluorescence correlation spectroscopy (FCS). In contrast to classical diffusion experiments, the single-molecule approach allowed for the direct determination of diffusion coefficients for lysozyme, trypsin inhibitor, BSA, and IgG both inside the hydrogel and after being released into the solution. The results of the FCS analyses and the calculated pristine in-gel diffusion coefficients were compared with the values obtained from the Stokes-Einstein equation, Fickian diffusion models, and the literature. The release kinetics suggested that protein diffusion through nanofiber hydrogels depended primarily on the size of the protein. Protein diffusivities decreased, with increasing hydrogel nanofiber density providing a means of controlling the release kinetics. Secondary and tertiary structure analyses and biological assays of the released proteins showed that encapsulation and release did not affect the protein conformation and functionality. Our results show that this biocompatible and injectable designer self-assembling peptide hydrogel system may be useful as a carrier for therapeutic proteins for sustained release applications. drug delivery ͉ protein diffusion ͉ single-molecule analysis ͉ spectroscopic analyses ͉ antibody-antigen interactions H ydrogels have long been recognized as being well suited for numerous biomedical applications, including regenerative medicine and controlled drug release (1-3). The successful implementation of these materials, however, depends on many factors, including component and degradation product toxicity, inflammatory host response, the ease of incorporating cellspecific bioactive moieties, and the controlled and sustainable release of the active compound over prolonged periods of time. Despite the intense research conducted on myriad natural and synthetic materials (i.e., polyglycolic-polylactic acid, agarose, collagen, alginate, etc.), all of these challenges have not been resolved yet for a single system (2, 4).In 1993, we discovered that a class of self-assembling peptides comprising alternating hydrophobic and hydrophilic amino acids spontaneously self-organize into interwoven nanofibers with diameters of 10-20 nm upon being introduced to electrolyte solutions (5). These nanofibers further organize to form highly hydrated hydrogels [up to Ϸ99.5% (wt/vol) water], with pore sizes between 5 and 200 nm in diameter. Peptide hydrogels not only have all of the advantages of ''traditional'' hydrogels but also do not use harmful materials (e.g., toxic cross-linkers, etc.) to initiate the solution-gel transformation (2) whereas the degradation products are natural amino acids, which can be metabolized. The fact that the solution-gel transition occurs at physiological conditions and the high internal hydration of the hydrogel allows for ...
