Category: Formulation and Quality
Purpose: Peptides are interesting therapeutic molecules with specific and potent action over a wide variety of diseases. Currently, most of them are parenterally administered resulting in poor patient compliance and important overall healthcare costs. Indeed, oral bioavailability of peptides is strongly limited by their proteolytic degradation in the duodenal lumen as well as their poor permeability across the intestinal epithelium. In this study, we considered encapsulation of peptides in biodegradable and biocompatible Nanostructured Lipid Carriers (NLC) and Solid Lipid Nanoparticles (SLN) to overcome both oral bioavailability limiting aspects.
The objective was to encapsulate Leuprolide (LEU), a hydrophilic model peptide, in NLC and SLN with a high Encapsulation Efficiency (EE) and in a reproducible and scalable manner. For this purpose, LEU was hydrophobized by the formation of a Hydrophobic Ion Pair (HIP). After characterization of the systems (size, morphology, EE), their behavior was evaluated in simulated gastro-intestinal fluids. Next, the capacity of the nanoparticles to protect LEU from proteolytic degradation by trypsin and α-chymotrypsin was evaluated in vitro. The ability of the lipid-based nanosuspensions to increase LEU permeability across the intestinal barrier was evaluated on Caco-2 cell model (enterocyte-like model). The effect of mucus on the transport was also studied, using Caco-2:HT29-MTX (goblet cells, mucin-secreting cells) co-cultures.
Methods: The lipophilicity of LEU was increased by formation of a HIP by reversible electrostatic interaction with sodium docusate. HIPs were encapsulated within the nanoparticles composed of Precirol®ATO5, Kolliphor®RH40 and CapryolTM90 in the case of NLC. Nanosuspensions were formulated by a melt High Pressure Homogenization (HPH) technique using a Microfluidizer LM 20 (Microfluidics).
EE was measured by quantifying free LEU in the supernatant by HPLC.
Particle size was evaluated by Dynamic Light Scattering and confirmed by cryo-TEM while assessing particles morphology. Particle size stability was evaluated in Simulated Gastric Fluid (SGF), Fasted State Simulated Intestinal Fluid (FaSSIF-V2) and Fed State Simulated Intestinal Fluid (FeSSIF-V2) at 37°C. Leuprolide release was evaluated in FaSSIF-V2. Degradation studies by trypsin and α-chymotrypsin were conducted in pH 6.8 buffer. The reaction was stopped at different time points and remaining LEU was quantified by HPLC.
To evaluate the ability of the nanoparticles to increase the permeability of LEU, formulations were co-incubated with Caco-2 or Caco-2/HT29-MTX cell monolayers. After 2 h, the basolateral medium was collected to further quantify the amount of transported LEU.
Uptake of the nanoparticles (containing HIP and a fluorescent dye) by Caco-2 was evaluated qualitatively by confocal microscopy. Following the transport study, cells were fixed in paraformaldehyde, stained and observed by confocal microscopy. The internalization of the nanoparticles by Caco-2 was quantified by flow cytometry after 2h co-incubation with the nanosuspensions.
Results: The HPH method enabled reproducible formulation of LEU-loaded NLC and SLN. The nanoparticles (blank, LEU or HIP-loaded) were platelet-shaped with a Z-average around 115 nm and a PDI of 0.2. A significant difference was observed between encapsulation of neat LEU (44% in NLC, 52% SLN) and HIP (76% in NLC, 78 in SLN), Figure 1.
No change in particle size nor PDI was observed upon dispersion of the nanosuspensions in simulated gastro-intestinal fluids. However, an important burst release was measured in FaSSIF-V2.
Encapsulation of LEU in NLC significantly improved its resistance to trypsin, which was not observed with SLN. However, both systems failed in providing protection towards α-chymotrypsin activity.
The confocal microscopy images have shown internalization of both systems by cell monolayers, which seemed higher for NLC than SLN. For Caco-2:HT29-MTX monolayers, particles were also observed, indicating the ability of the formulations to cross the mucus barrier, Figure 2. Flow cytometry results were in accordance with these observations. Indeed, the mean fluorescence for SLN was 1582 a.u., significantly different from the one measured with NLC: 4558 a.u. These values corresponded to 81% Caco-2 cells responding positive to SLN and 98% cells for NLC.
Conclusion: This study has shown the possibility to reproducibly encapsulate a hydrophilic peptide in solid lipid-based nanocarriers with an important EE using the HIP-formation technique.
Despite an important release observed in FaSSIF-V2, particles were stable in simulated gastro-intestinal media. Moreover, degradation studies have revealed the ability of NLC to significantly prolonged leuprolide integrity in vitro.
The interaction studies between Caco-2 and the formulations have shown an important uptake of particles, especially in the case of NLC. These data indicate promising results regarding permeability improvement of leuprolide across the intestinal epithelium.
The in vitro evaluation of the systems would suggest an improvement of leuprolide oral bioavailability after encapsulation in solid lipid nanocarriers.
Vincent Jannin– Manager ODDI, Lonza, Illkirch-Graffenstaden Cedex, Alsace, France
Ana Beloqui– Woluwe-Saint-Lambert, Brussels, Belgium
Veronique Preat– Woluwe-Saint-Lambert, Brussels Hoofdstedelijk Gewest, Belgium
Hatem Fessi– Villeurbanne, Rhone-Alpes, France
Sandrine Bourgeois– Villeurbanne, Rhone-Alpes, France