Effect of Lipid Composition on the Biopharmaceutical Properties of Exenatide-Loaded Lipid Nanoparticles for Oral Delivery

Oral delivery of peptides is limited by extensive degradation and poor absorption in the gastrointestinal tract. Lipid nanoparticles (LNPs) have emerged as promising platform to enhance the stability and intestinal absorption of therapeutic peptides. In the present work, we investigated the influence of phospholipids with different transition temperatures DOPC (−5 °C), EPC (18 °C), and HSPC (77 °C) on the biopharmaceutical properties of phospholipid-based LNPs for the oral administration of exenatide, used as a model GLP-1 peptide. Exenatide-loaded LNPs were produced by microfluidics using hydrophobic ion pairing (HIP) with the cationic lipid DOTAP to promote peptide encapsulation. The DOTAP/exenatide complex was efficiently incorporated into the lipid matrix, yielding high encapsulation efficiencies (~95%) and loading capacities (~10%). Post-assembly surface modification with DSPE-PEG2kDa at two PEGylation levels (10% or 30% w/w) was introduced to shield the positive zeta potential and modulate diffusion across intestinal mucus. The resulting LNPs exhibited sizes of approximately 80-120 nm, low polydispersity (PDI < 0.2), and a positive surface charge (~+35 mV), which decreased following PEGylation. Membrane fluidity assessed by DPH fluorescence anisotropy indicated that LNPDOPC displayed the highest fluidity (4.2), followed by LNPEPC (3.9), whereas LNPHSPC exhibited the lowest fluidity (3.5). Laurdan generalized polarization measurements showed a similar trend, confirming the differences in matrix rigidity among formulations. Mucus diffusivity studies using FRAP demonstrated significantly lower fluorescence recovery for non-PEGylated LNPs, ranging from approximately 30 to 50%, indicating reduced nanoparticle mobility across the mucus network. In contrast, PEGylated formulations exhibited enhanced fluorescence recovery, reaching 55-63% with 10% PEG and 60-72% with 30% PEG. This improvement was attributed to the partial shielding of surface cationic charges by PEG, which reduced electrostatic interactions with negatively charged mucin components and promoted mucus penetration. Cellular association studies in a mucus-producing Caco-2/HT29-MTX co-culture model showed that increasing PEG surface density from 10 to 30% progressively reduced LNP association with both the mucus and cellular layers. Among the PEGylated formulations, 10% PEGylation appeared to provide a favorable balance between mucus diffusivity and cellular association. Notably, DOPC- LNPs showed greater localization within both mucus and cellular compartments than EPC- and HSPC-formulations, consistent with their enhanced mobility observed in FRAP studies. Permeability studies on the same model indicated that 10% PEGylation provided the best balance between mucus penetration and epithelial permeability across all formulations, particularly for LNPDOPC, likely due to their greater membrane flexibility. Overall, these findings highlight the potential of PEGylated phospholipid-based LNPs as a platform for the oral delivery of exenatide and other peptide therapeutics, while demonstrating that phospholipid composition and PEGylation level can be used to modulate key properties such as membrane fluidity, mucus diffusivity, cellular association, and intestinal permeability.