Permeation of unfolded basic fibroblast growth factor (bFGF) across rabbit buccal mucosa--does unfolding of bFGF enhance transport?

PURPOSE: To investigate whether recombinant human basic fibroblast growth factor (rhbFGF) would permeate freshly-excised rabbit buccal mucosa. In addition, the effect of a permeation enhancer (Na+ glycocholate) and the possibility of reversibly unfolding the globular protein to a more linear conformation to increase the permeability of the test protein was evaluated.
METHODS: The in vitro flux of bFGF through freshly-excised rabbit buccal mucosa was determined using side-by-side diffusion systems. Detection of bFGF was performed using gradient elution, reversed-phase high-pressure liquid chromatography (RP-HPLC). Fluorescence spectroscopy and heparin affinity chromatography were used to assess the tertiary structure of bFGF.
RESULTS: Preliminary in vitro results have demonstrated that the bFGF flux increased from 1.4 +/- 0.13 ng min-1 cm-2 to 3.2 +/- 0.38 ng min-1 cm-2 with the addition of 15 mM Na+ glycocholate (NaG) to the donor solution. Subsequent addition of guanidine HCl (GnHCl) to the donor solution (3 M) was not followed by a further increase in the flux of bFGF (2.9 +/- 0.26 ng min-1 cm-2). However, when the order of addition of the additives was reversed (GnHCl first followed by NaG), the flux of bFGF across rabbit buccal mucosa was increased. Upon addition of GnHCl, there was a significant (p < .05) increase in bFGF flux from 1.2 +/- 0.15 ng min-1 cm-2 to 5.0 +/- 0.58 ng min-1 cm-2. Addition of NaG further increased the flux to 8.5 +/- 1.1 ng min-1 cm-2 which was approximately 3- to 3.5-fold greater than that determined with the protein alone in the absence of any donor phase additives. The percent of parent bFGF remaining following a 3-hr exposure of a bFGF solution to either the mucosal, serosal, or both sides of rabbit buccal mucosa were 54.3 +/- 5.7%, 71.8 +/- 6.3%, and 36.2 +/- 5.4%, respectively with the majority of parent bFGF lost during the first 15 minutes. A model endopeptidase (endoproteinase Arg-C from mouse submaxillary gland) was shown in vitro to contribute to the loss in parent bFGF.
CONCLUSIONS: The permeation of bFGF across rabbit buccal mucosa may be significantly increased by initially unfolding the protein with GnHCl and then treating the tissue with the permeation enhancer, NaG. Refolding and possible reactivation of bFGF's bioactivity may occur following membrane transport and subsequent dilution into an infinite sink.