Formulation of Peptide Loaded Layer-by-Layer Nanoparticles Targeting PepT1 Intestinal Transporters: A new strategy for oral peptide delivery

B. Gourdon, C. Chemin, J.-M. Péan, P.T. Hammond
Massachusetts Institute of Technology,
United States

Keywords: oral delivery, peptide, nanoparticles, layer-by-layer


While peptide based therapeutics have been developed significantly in the past decades, delivery challenges have limited their clinical use. Although oral delivery is preferred, conventional formulation strategies cannot be applied due to limited oral bioavailability of these new molecular entities. The low bioavailability of these compounds is directly linked to their poor ability to reach the systemic blood circulation. One current strategy to improve oral bioavailability is targeting intestinal transporters, such as PepT1. Its broad substrate specificity makes it an attractive transporter to target. By modifying a drug having a low oral bioavailability with a peptide-like ligand, it allows its intestinal transport by PepT1 into the bloodstream. Targeting PepT1 with prodrugs has been widely studied, and one well-known commercial example Valacyclovir improved the oral bioavailability of the antiviral Acyclovir up to 5-fold in humans. Recently, it has been evidenced that intestinal PepT1 transporters can be targeted with valine functionalized polylactic acid–polyethylene glycol nanoparticles (NPs). This strategy showed a significant 2-fold increase of the apparent permeability of the encapsulated oxytocin peptide in vitro in Caco-2 cells compared to free drug. In order to translate this strategy to the clinic, controlling peptide release from NPs through the gastro intestinal tract is crucial. To achieve a well-controlled release, it was proposed to employ Layer-by-Layer (LbL) assembly to carefully tune drug release and enhance intestinal drug absorption. LbL is a powerful technique to create multifunctional NPs. Nanoscale layers of biocompatible polyelectrolytes are added via iterative electrostatic adsorption in a simple, water based process. Importantly, LbL assembly will provide functional NPs with the ability to control peptide release while targeting intestinal PepT1 transporter. In this work, a candidate peptide was first encapsulated into negatively charged liposomes. NPs were then layered successively with three polyelectrolytes: a positively charged polymer selected for its controlled release properties, then a negatively charged polymer, and, finally, an outer positively charged polymer functionalized with valine in order to target intestinal transporter PepT1. NP synthesis is set up so as to get particles under 200 nm in diameter allowing them to cross the intestinal mucus barrier, and the surface charge was monitored at each layering step to maintain zeta potential>30 mV to assure the stability of the NPs. Using flow cytometry and structured illumination microscopy experiments, NP uptake and intracellular location were assessed qualitatively and quantitatively in a Caco-2 cell culture model. Co-incubation with inhibitory concentrations of glycylsarcosine, a well-known substrate of PepT1, decreased NP uptake, confirming transporter targeting. Peptide release was optimized such that 30 % of drug was released over 2 hours, allowing time for NPs to engage with PepT1 and thus be processed by intestinal epithelial cells. Finally, drug transport experiments will evaluate peptide transport across a model of the intestinal membrane, and will be compared non-LbL particles and free compound, revealing the differential effects of LbL components and PepT1 targeting on apparent permeability. Oral administration of peptide-based therapeutics remains a challenging issue. Bringing together strategies of tissue-specific targeting and LbL assembly, it is possible to develop innovative formulations.