DEVELOPMENT OF ENGINEERED SOLID LIPID NANOPARTICLES BY MICROFLUIDICS FOR ORAL DELIVERY OF PEPTIDES

In this project, solid lipid nanoparticles were selected for delivery of a model peptide via oral administration. Microfluidics technology was employed for the preparation of the SLNs in order to obtain a reproducible and scalable formulation process In previous studies, the composition and setting for microfluidic process have been extensively investigated. SLNs were formulated by processing a lipid mixture 1 : 3 of HSPC and cholesterol in ethanol; while in the aqueous phase, the peptide was precomplexed with a cationic lipid, DOTAP, in a ratio of 1:12 peptide / DOTP mola ratio, in 1mM HEPES buffer at pH 8. The use of Tri-poly-phosphate was the first surface decoration method used, but no significant and useful results were obtained for the purposes of the project. The cation charge of the SLNs was masked by a post-insertion decoration with DSPE-mPEG at 2-30% (w / w) in order to obtain particles with a charge close to neutrality, so as to avoid interactions with the mucus in the intestinal environment. Concentrations of 10 and 30% w / w were selected for size, high PEG density and low zeta potential. TEM imaging showed how the spherical morphology of the SLNs was kept intact even during pegylation and the size is in agreement with the results from the DLS analysis. Fluorescence recovery after photosbleaching (FRAP) analysis was employed to investigate the motility of SLNs in mucin; the results show that pegylation positively modulates diffusion, reducing the interaction with mucin, which vice versa the cationic charge entails. The colloidal stability of SLNs in SIF and culture medium shows how the stealth effect of pegylation increases stability and prevents opsonization. SLNs were then converted to lyophilized powder in order to be loaded into eneteric-coted capsules. Trehalose and mannitol have been identified as preferential cryoprotectants for maintaining particle size quality and for obtaining a good cake, respectively. For this purpose, a lyoprotectant mixture of trehalose and mannitol 1: 1 w / w at a total of 2% (w / v) concentration was selected. Mass spectroscopy analysis and circular dichroism studies were used to confirm the stability and integrity of the chemical structure of the peptide, during all steps of the formulation process. Cellular studies on human intestinal Caco-2 cells were used to simulate the intestinal environment. Biocompatibility studies of SLNs decorated with 0, 10, and 30% w / w DSPE-PEG show a safe profile with cell viability above 80% under all conditions. As hypothesized, charge neutralization with pegylation to reduce interaction with cells was confirmed by cell association studies with flow cytometric analysis and confocal microscopy. Results show over 50% association efficiency of the not PEGylated SLNs while the 10 and 30 percent w / w DSPE-PEG2kDa coating decreased the cell association