Category: Formulation and Quality
Purpose: While peptide- and protein-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 because of limitations to cross the intestinal epithelium, degradation in the gastrointestinal tract and their large molecular size [Johnson,1994; Kwan,1997]. One current strategy to improve oral bioavailability is targeting intestinal transporters, such as PepT1. This transporter is well known to promote the uptake of dipeptides, tripeptides and peptidomimetics due to an inward proton gradient. 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 the well-known commercial example Valacyclovir improved the oral bioavailability of the antiviral Acyclovir by 3- to 5- fold in humans [Beauchamp,1992]. Recently, it has been evidenced that intestinal PepT1 transporters can be targeted with valine functionalized polylactic acid–polyethylene glycol (PLA-PEG) nanoparticles (NPs) [Gourdon,2017,2018]. 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 gastrointestinal 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 [Hammond,2012; Correa,2016]. Importantly, LbL assembly will provide functional NPs with the ability to control peptide release while targeting intestinal PepT1 transporter.
Methods: In this work, a candidate peptide, usually administered subcutaneously for the treatment of type 2 diabetes, was first encapsulated into negatively charged liposomes. These 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 [Ensign,2012], 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, was carried out in order to confirm transporter targeting. Peptide release was optimized, allowing time for NPs to engage with PepT1 and thus be processed by intestinal epithelial cells. Finally, drug transport experiments evaluated peptide transport across a model of the intestinal membrane, and allowed to compare LbL particles and free compound, revealing the differential effects of LbL components and PepT1 targeting on apparent permeability. Future in vivo experiments on diabetic db/db mice will allow to determine the pharmacokinetic profile of the candidate peptide according to its way of administration (intravenously, subcutaneously or orally) and its formulation (free, loaded in LbL or bare liposomes). Finally, blood glucose level measurements over time will confirm the efficacy of our strategy.
Results: The candidate peptide was passively loaded into liposomes and 140 nm NPs were obtained before layering. Addition of three polyelectrolytes layers increased the size up to 187 nm and reversed zeta potential from -59 mV to 57 mV. Drug loading and encapsulation efficiency were 7.44% and 29.75%, respectively, and a good control of the release was observed as only 60% of the drug was released after 2 hours for layered NPs compared to 85% for bare NPs (Fig. 1). In vitro , flow cytometry experiments showed a significant 1.5 times increase of the median fluorescent intensity when Caco-2 cells were incubated with valine functionalized NPs compared to bare liposomes. This fluorescence decreased with the increase of glycylsarcosine in competition, confirming PepT1 transporter targeting (Fig. 2). Finally, structured illumination microscopy of the cells after incubation with Val-NPs, showed a localization of the NPs on the membrane (Fig. 3). Drug transport experiments have been carried out on Caco-2 cells seeded on Transwells in order to assess the output of peptide encapsulation in LbL NPs compared to free drug. Results analysis is ongoing.
Conclusion: Oral administration of peptide- and protein-based therapeutics remains a challenging issue. Bringing together strategies of tissue-specific targeting and Layer-by-Layer assembly, it is possible to develop innovative formulations and identify a candidate with translational potential.