Identification of Novel Medulloblastoma Cell-Targeting Peptides for Use in Selective Chemotherapy Drug Delivery

Abstract

Medulloblastoma is a malignant brain tumor diagnosed in children. Chemotherapy has improved survival rates to approximately 70%; however, children are often left with long-term treatment side effects. New therapies that maintain a high cure rate while reducing off-target toxicity are required. We describe for the first time the use of a bacteriophage-peptide display library to identify heptapeptides that bind to medulloblastoma cells. Two heptapeptides that demonstrated high [E1-3 (1)] or low [E1-7 (2)] medulloblastoma cell binding affinity were synthesized. The potential of the peptides to deliver a therapeutic drug to medulloblastoma cells with specificity was investigated by conjugating E1-3 (1) or E1-7 (2) to doxorubicin (5). Both peptide−drug conjugates were cytotoxic to medulloblastoma cells. E1-3 doxorubicin (3) could permeabilize an in vitro blood− brain barrier and showed a marked reduction in cytotoxicity compared to free doxorubicin (5) in nontumor cells. This study provides proof-of-concept for developing peptide−drug conjugates to inhibit medulloblastoma cell growth while minimizing offtarget toxicity.

Modulating the Selectivity and Stealth Properties of Ellipsoidal Polymersomes through a Multivalent Peptide Ligand Display

“…We previously reported a detailed characterization of the targeting peptide (FSPRPAFL) used for functionalizing these polymersomes and showed that the peptide is capable of mediating a ligand-mediated endocytosis. [21] Herein we confirm that such metabolically active mechanism was also responsible for the transport of the polymersomes into the target cells. In brief, DAOY cells were seeded onto tissue culture plates and incubated with 0.025 mg/mL of low-ligand density polymersomes at 37 °C and 4 °C for 2 hours (concentration was chosen according to the maximum tolerated concentration tested, see Supporting Information Figure S8).…”

“…Heptapeptide FSRPAFL, which was previously described to have selectivity towards our target medulloblastoma cell line (DAOY), [21] was conjugated onto the PEG43-b-P(NIPAM21-co-t-BocAEA9-co-PFP4) polymer backbone using a diisopropylethylamine base in N,Ndimethylformamide for 48 hours at 40 °C. To synthesize the lower ligand-density (LLD) polymer, only 1.5 molar equivalent of this peptide was reacted onto the polymer backbone, which contains 4 repeating units of pentafluorophenol -as indicated by the 1 H NMR (Scheme S2, Supporting Information).…”

“…After removal of the polymersomes, the cells were washed with phosphate saline buffer, trypsinized and harvested for flow cytometry analysis using methods previously described. [21] The fluorescent intensities were corrected for the differences observed in each sample (Figure S9, Supporting Information).…”

“…PEG43-b-P(NIPAM21-co-t-BocAEA9-co-PFP4) (300 mg, 1 equiv. ), FSRPAFL peptide [21] (36 mg, 1.5 equiv. ), and diisopropylethylamine (100 µL, excess) were dissolved in peptide-grade N,N-dimethylformamide (4 mL) and stirred for 2 days at 40 ˚C under nitrogen flow.…”

“…These polymersomes were functionalized with a heptapeptide, FSRPAFL which we discovered through a peptide phage-display library as having selectivity and high binding affinity against human medulloblastoma (DAOY) cells. [21] The stealth property of these ellipsoidal polymersomes was determined through their association and clearance by human phagocytes and compared against a known "stealthy" PEG-based particle by incubating the particles with whole human blood. [22] We hypothesized that the higher peptide ligand density will give greater selectivity to these polymersomes and that the peptide will not alter their-like stealth property.…”