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 gro wth factor (rhbFGF) would permeate freshly-excised rabbit buccal mucos a. 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 pro tein was evaluated. Methods. The in vitro flux of bFGF through freshly -excised rabbit buccal mucosa was determined using side-by-side diffus ion systems. Detection of bFGF was performed using gradient elution, r eversed-phase high-pressure liquid chromatography (RP-HPLC). Fluoresce nce spectroscopy and heparin affinity chromatography were used to asse ss the tertiary structure of bFGF. Results, Preliminary in vitro resul ts 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 additi on of guanidine HCl (GnHCl) to the donor solution (3 hi) was not follo wed 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 rev ersed (GnHCl first followed by NaG), the flux of bFGF across rabbit bu ccal mucosa was increased. Upon addition of GnHCl, there was a signifi cant (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 increase d 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 th e absence of any donor phase additives. The percent of parent bFGF rem aining following a 3-hr exposure of a bFGF solution to either the muco sal, 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 pa rent bFGF lost during the first 15 minutes. A model endopeptidase (end oproteinase 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 in itially 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 subs equent dilution into an infinite sink.