Description
The oral delivery of biological drugs remains a major pharmaceutical challenge due to the extensive degradation of biomolecules within the gastrointestinal tract (GIT).
To overcome these challenges, lipid nanoparticles (LNPs) have recently emerged as a promising platform to enhance the stability and absorption of therapeutic peptides.
Herein, the influence of three phospholipids, EPC, DOPC, and HSPC, on the biopharmaceutical performance of LNPs for oral administration of the exenatide, a GLP-1 analog for the treatment of Type II Diabetes Mellitus, was investigated. LNPs were produced using the NanoAssemblrTM system (Precision NanoSystems Inc., Vancouver, Canada) employing staggered herringbone micromixer (SHM) cartridges. The selection of the Flow Rate Ratio (FRR) and Total Flow Rate (TFR) was performed through an extensive screening process conducted by our research group1. The organic phase consisted of each phospholipid combined with cholesterol in a 1:3 molar ratio, while the aqueous phase contained exenatide pre-complexed with the cationic lipid DOTAP at a 1:12 molar ratio, through hydrophobic ion-pairing (HIP). The peptide feed was fixed at 10% (w/w) relative to the total lipid mass. The DOTAP/exenatide complex was efficiently incorporated into the lipid matrix, yielding high encapsulation efficiencies around 95% and loading capacities, around 10%.
Physical characterization via Dynamic Light Scattering (DLS) showed particle sizes of ~90 nm for LNPEPC and LNPDOPC and ~120 nm for SLNHSPC, all with PDI values <0.2 and positive zeta potentials around +40 mV for LNPEPC and LNPDOPC and +50 mV for SLNHSPC, confirming the incorporation of the cationic lipid DOTAP.
Post-assembly surface modification with DSPE-PEG2kDa, with two degrees of PEGylation, (10% or 30% w/w) markedly reduced the zeta potential, to ~20 mV and <10 mV for both LNPEPC and LNPDOPC and ~15 mV and -3 mV for SLNHSPC, while maintaining colloidal stability.
All formulations demonstrated good colloidal stability in the presence of lipase, simulated gastric fluid (SGF), and simulated intestinal fluid (SIF), which mimic the physiological gastrointestinal environment.
Drug-release studies, conducted in SGF and SIF, revealed sustained release for up to 96 hours in PEGylated LNPEPC and LNPDOPC, compared to complete release within 72 hours for non-PEGylated counterparts. Non-PEGylated SLNHSPC exhibited complete peptide release after 10 days, while PEGylated counterparts reached respectively 93% and 84% at the same time.
Enzymatic stability assays, conducted for two hours in SGF with pepsin followed by the following 6 hours in SIF with trypsin, showed enhanced peptide protection by PEGylated LNPs, which retained ~65% of exenatide versus ~50% in non-PEGylated counterparts.
Cellular association and permeability studies demonstrated that increasing PEG surface density reduced nanoparticle interaction with cells.
Furthermore, confocal microscopy of co-culture models (Caco-2/HT29-MTX) provided preliminary evidence of LNP colocalization within the intestinal epithelium.
Citations:
1. Büşra Arpaç Birro, Cristiano Pesce, Francesco Tognetti, Agnese Fragassi, Lisa Casagrande, Mariangela Garofalo, Stefano Salmaso, Paolo Caliceti, et al. Unlocking the potential of microfluidic assisted formulation of exenatide-loaded solid lipid nanoparticles, International Journal of Pharmaceutics, Volume 678, 2025, 125686, ISSN 0378-5173