(558f) Esterified Peptide Prodrugs for Nanocarrier Hitchhiking

While there are many promising synthetic and natural carriers for peptide therapeutics, most rely on hydrophobic or electrostatic interactions to encapsulate their cargo. However, encapsulating hydrophilic peptides with low net charge into these carriers is difficult. For protein therapeutics, altering amino acids in the protein sequence or adding highly charged peptide tags assists with encapsulation; yet, altering the sequence of smaller peptides can have a significant effect on therapeutic peptide activity. Here, we describe a strategy in which we reversibly modulate the hydrophobicity and charge of therapeutic peptides by capping anionic carboxylates with hydrophobic esters. One such therapeutic is the cardioprotective peptide αCT11 (RPRPDDLEI), which has 4 carboxylic acid groups and several arginine groups that render it highly hydrophilic; for example, capping all of the anionic carboxylic acids on αCT11 increases its net charge from -1 to +3 while also increasing its hydrophobicity, as shown by high performance liquid chromatography (HPLC) elution time. We developed a prodrug scheme in which esters are installed on therapeutic peptides to temporarily increase their hydrophobicity/net charge and promote encapsulation into nanocarriers. Once administered, native ester cleaving-enzymes provide an opportunity to cleave the installed esters and revert the therapeutic peptides to their active form. We synthesized esterified versions of αCT11, with 1-4 esters, in high purity (> 95%), as confirmed by HPLC and electrospray ionization mass spectrometry. Using HPLC, we assessed the hydrophobicity, hydrolysis kinetics, and encapsulation of esterified αCT11 derivatives. Peptide retention time, reflective of hydrophobicity, increased with the number of esterified carboxylic acids on αCT11. Under basic conditions that accelerate hydrolysis, both the number and type of installed esters were found to affect hydrolysis kinetics. For example, αCT11 with one allyl ester hydrolyzes to its active form faster than αCT11 with 4 methyl esters, with pseudo-first order rate constants 8.61 x 10^-7 and 4.17 x 10^-7 s^-1, respectively. We are encouraged by preliminary data showing the encapsulation of αCT11 with 1 allyl ester into exosomes, natural vesicular carriers that are particularly relevant, as they contain ester cleaving-enzymes. In the future we look forward to leveraging the precision of peptide synthesis to further relate the structure of esterified peptide prodrugs (e.g., varying the number, position, and type of ester) to their encapsulation and subsequent activation. In summary, the work presented herein shows the potential of peptide esterification as a prodrug scheme to broaden the benefits of encapsulation to hydrophilic peptide therapeutics with low net charge.