Synthesis and Characterization of CREKA Peptide-Conjugated Micelles

The [Cys–Arg–Glu–Lys–Ala] peptide was synthesized using standard Fmoc-mediated solid phase peptide synthesis methods on rink Amide resin (Anaspec, Fremont, CA, USA) using an automated PS3 Benchtop Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA). Peptides were cleaved and deprotected with 94:2.5:2.5:1 by volume trifluoroacetic acid: 1,2-ethanedithiol:H₂O:triisopropylsilane and were precipitated and washed several times with cold diethyl ether, dissolved in water, lyophilized, and stored as lyophilized powders at 20 °C. Crude, peptide mixtures were purified by reverse-phase HPLC (Prominence, Shimadzu, Columbia, MD, USA) on a C8 column (Waters, Milford, MA) at 55 °C using 0.1% trifluoroacetic acid in acetonitrile/water mixtures and characterized by MALDI-TOF/TOF mass spectral analysis (Biflex III, Bruker, Billerica, MA, USA). Cysteine-containing peptides were conjugated via a thioether linkage to DSPE-PEG(2000)-maleimide (Avanti Polar Lipids, Alabaster, AL, USA) by adding a 10% molar excess of the lipid to peptide in water. After reaction at RT for 24 h, the resulting product was purified and characterized as described above. Cy7 was conjugated via a peptide bond to DSPE-PEG(2000)-amine (Avanti Polar Lipids, Alabaster, AL, USA) by adding an equal molar equivalent of Cy7 mono-N-hydroxysuccinimide ester (GE Healthcare Life Sciences, Pittsburgh, PA, USA) to the lipid dissolved in 10 mM aqueous sodium carbonate buffer (pH 8.5). After reaction at RT for 24 h, the mixture was also purified on a C4 column and characterized as described above.
CREKA micelles were assembled by dissolving the Cy7 and peptide containing DSPE-PEG(2000) amphiphiles in methanol (10:90 molar ratio), mixing the components, and evaporating the mixed solution under nitrogen. The resulting film was dried under vacuum O/N, and then hydrated at 80 °C for 30 min in water or PBS and allowed to cool to RT. Non-targeting micelles were assembled by combing DSPE-PEG(2000)-Cy7 and DSPE-PEG(2000)-maleimide.