M. Barlek1, D. C. Gillis1, S. A. Egner2, S. I. Stupp2, N. D. Tsihlis1, M. R. Karver2, M. R. Kibbe1 1University Of Virginia, Charlottesville, VA, USA 2Northwestern University, Chicago, IL, USA
Introduction: Peptide amphiphile (PA) nanofibers have been shown to target and deliver drugs when delivered systemically through an intravenous (IV) injection. However, IV administration is cumbersome and requires a skilled practitioner. Subcutaneous (SC) administration of medications has multiple advantages over IV administration. However, successful uptake of drugs from the SC space requires specific characteristics. Residues in the β-sheet-forming region of PAs can be modified to tune β-sheet strength and the overall charge. As such, the aim of our study was to identify PA nanofibers that can be absorbed into the systemic circulation after SC administration. We hypothesized that negatively charged PA nanofibers with weak β-sheet formation would be absorbed systemically the best following SC injection.
Methods: Four PAs were selected based on amino acid charge and propensity to form β-sheets: two net positive and two net negative charged PAs, each with strong and weak β-sheet forming regions. PAs were co-assembled at a molar ratio of 95% unlabeled PA to 5% PA labeled with IR750 dye. Transmission electron microscopy imaging was used to confirm nanofiber formation. Secondary structure analysis was performed by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). Male and female Sprague Dawley rats (12 wk) were injected in the dorsum of the neck with PA (5 mg). In vivo normalized abdominal fluorescence was measured 1-72 hours after injection using a LagoX in vivo imaging system. Statistical analysis was performed using a two- or three-way ANOVA.
Results: Examination of secondary structure by CD and FTIR supported that both positive (+3) and negative (-1) charge PAs containing residues predicated to form strong β-sheets were primarily β-sheet in character. Positive and negative charge PAs containing weak β-sheet forming regions showed mixed secondary structure. The PA that was negatively charged with a weak β-sheet region had the highest amount of abdominal fluorescence at 1 hour (2.5×10-3cm 2) and had significantly higher abdominal fluorescence among all PAs at 6 hours (6.2×10-3cm 2) and 24 hours (7.1×10-3cm 2) after SC injection (p<0.05). For all PAs, abdominal fluorescence peaked at 24 hours after injection and sharply declined at 48 hours, with no significant difference between the PAs at 72 hours (p=ns). No significant difference was found in abdominal fluorescence between male and female rats (p=ns). Ex vivo imaging of organs at 72 hours showed that PAs were only detected in the liver and the kidney.
Conclusion: Our data shows that negatively charged PAs with weak β-sheet forming regions had the greatest ability to be absorbed into the systemic circulation following SC administration. No difference in systemic absorption was noted by sex. This work lays the foundation for the design and development of targeted nanotherapeutics that can be systemically administered through SC injections.