We report on the synthesis, kinetics of proteolysis by trypsin, and morphological characterization of a
novel lipidated decapeptide that spontaneously self-assembles in aqueous solutions into 0.5 μm diameter
hollow tubules and helices that range in length from tens to hundreds of micrometers depending on formation
conditions. We also report on an improved method for the tritioacetylation of peptides. Tight molecular
packing of the peptide−amphiphile into a crystalline bilayer array forces tight packing between peptide
headgroups, which was found to significantly protect the peptides from proteolysis by trypsin. Relief of
this steric hindrance between peptide headgroups caused by solubilization of the bilayer into detergent
micelles accelerated the rate of trypsin hydrolysis by 32,000-fold. Raman spectroscopy and circular dichroism
spectropolarimetry were used to gain molecular-level insight into the difference between hydrolysis rates.
Results obtained from these studies suggest that differences in molecular packing and conformation of the
peptide headgroups in crystalline tubular and dispersed micellar phases determine the extent of proteolytic
protection. Protection from proteolysis is considered a useful feature of lipidated peptide tubules for their
potential use as a depot of bioactive peptides and other labile prodrugs at defined biological sites for
sustained release.
A growing number of amphiphiles are known to form high axial ratio microstructures (HARMs) such as the hollow cylindrical microstructures called lipid tubules. As a prelude to exploring the potential of HARMs formed from lipopeptides in controlled release drug delivery, several microstructure formation conditions were investigated. We report the preparation of several glutamic acid dialkyl amides with varying alkyl chain lengths bearing a verity of peptides (1-4 amino acids) [peptide-Glu-(NHCnH2n+1)2, n=12, 14, 16]. These surfactants have been rapidly and efficiently converted into HARMs in aqueous buffer at physiological pH and ionic strength, or in buffer containing MeOH or EtOH. Helical ribbons and tubular HARMs were produced that were stable for as long as 6 months below the phase transition temperatures of the compounds. To estimate the stability of HARMs in vivo, HARMs formed from (Pro)3-Glu(NHC16H33)2 were incubated with DOPC liposomes or fetal calf serum at 40 degreesC. HARM size and shape did not change significantly, suggesting that such lipopeptide particles can retain their morphology long enough in vivo to be useful as drug delivery vehicles.
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