Design of lipid-based formulations for oral delivery of a BASP1 peptide targeting MYC-dependent gastrointestinal cancer cells

Abstract

Hypothesis

Oral delivery of the proliferation-inhibiting brain acid-soluble protein 1 effector domain peptide (Myr-NT) towards MYC-dependent gastrointestinal tumors can be achieved by forming hydrophobic ion pairs (HIPs) and incorporating them into lipid-based formulations.

Highlights

BASP1 effector peptide was encapsulated into a nanoemulsion via HIP formation.

Formulation boosted cellular uptake and limited enzymatic degradation of the cargo.

Formulation showed strong anti-proliferative effects in MYC-dependent cancer cells.

MYC-dependent gastrointestinal tumors are a suitable target for the delivery system.

Experiments

Hydrophobic ion pairing of fluorescently-labelled Myr-NT (Myr-NT-TAMRA) was performed, increase in lipophilicity was assessed, and the most promising HIP was subsequently incorporated into a nanoemulsion. Stability of the peptide towards degradation by trypsin was evaluated. Anti-proliferative and anti-invasive measurements were performed upon application of the loaded nanoemulsion on various MYC-dependent human cancer cell lines. Cellular uptake and molecular effect were complementary investigated by confocal laser scanning microscopy (CLSM) and by immunoblot analyses, respectively.

Findings

HIPs of Myr-NT-TAMRA exhibited up to 10,000-fold increase in lipophilicity, thereby enabling incorporation into a nanoemulsion. The formulation significantly boosted stability of incorporated peptide towards enzymatic degradation by trypsin. Furthermore, anti-proliferative measurements on human cancer cell lines revealed superior biological activity of the loaded nanoemulsion compared to the native peptide particularly in lymphoma cells, but also in colorectal cancer cells. Thereby, a correlation with proliferation inhibition as well as differences in MYC protein expression were observed. Finally, CLSM imaging revealed up to 15-fold increased cellular uptake of Myr-NT-TAMRA from the nanoemulsion confirming efficient intracellular delivery of the peptide.

Conclusion

Myr-NT can be efficiently delivered into intestinal tumor cells using orally administered lipid-based formulations.

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Materials

Fluorescently-labelled Myr-NT (myristoyl-GGKLSKKKKGK-TAMRA-COOH, Mr = 1780.6) and the unlabelled Myr-NT peptide (myristoyl-GGKLSKKKKG-OH, Mr = 1240.3) were commercially synthesized by the company PANATecs (Heilbronn, Germany). Lumogen Yellow (LGY) was a gift from BASF (Ludwigshafen, Germany). Lyso-Phosphatidylcholin was a gift from Lipoid GmbH (Ludwigshafen, Germany). Glyceryl monocaprylate (trade name: Capmul MCM C8) was provided as a free sample from Abitec Corporation (Columbus, OH, USA). Propylene glycol monocaprylate (trade name: Capryol 90) was a free sample from Gattefossé SAS (Lyon, France). Propylene glycol dicaprylate/dicaprate (trade name: Miglyol 840) was kindly donated by IOI Oleo GmbH (Hamburg, Germany).

Human erythrocyte concentrate was kindly provided by Tirol Kliniken GmbH (Innsbruck, Austria). Docusate sodium (DS) and soybean oil were purchased from Alfa Aesar (Kandel, Germany). Opti-MEM was obtained from Thermo Fisher Scientific (Vienna, Austria). Hoechst 33528 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Acetonitrile (ACN), calcium chloride (CaCl2), dimethyl sulfoxide (DMSO), glucose anhydrous, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), isopropylmyristate, methanol, octanol, PEG (20) – sorbitan monooleate (trade name: Tween 80), PEG (35) – castor oil (trade name: Kolliphor EL), potassium chloride (KCl), sodium chloride (NaCl), sodium dodecyl sulfate (SDS), sodium dodecyl benzene sulfonate (SDBS), trifluoroacetic acid (TFA), tris(hydroxymethyl)aminomethane (TRIS), trypsin from porcine pancreas (0.1 mg/mL) and Triton X-100 were purchased from Sigma-Aldrich (Vienna, Austria).

DPBS and the cell culture media DMEM, RPMI, and Leibovitz’s L-15 medium were purchased from PAN-Biotech (Aidenbach, Germany). Trypsin-EDTA (0.25 %) and glutamine were purchased from Life Technologies (Carlsbad, CA, USA), and Eximus Fetal Bovine Serum (FBS) from Catus Biotech (Tutzing, Germany). Glutaraldehyde was obtained from Merck (Darmstadt, Germany), and acrylamide (Rotiphorese) from Carl Roth (Karlsruhe, Germany).

Dennis To, Christian Steinbring, Leonie I. Weber, Fabrizio Ricci, Ilaria Polidori, Annika Postina, Markus Hartl, Andreas Bernkop-Schnürch, Design of lipid-based formulations for oral delivery of a BASP1 peptide targeting MYC-dependent gastrointestinal cancer cells, Journal of Controlled Release, Volume 382, 2025, 113677, ISSN 0168-3659, https://doi.org/10.1016/j.jconrel.2025.113677.