Development and characterization of peptide loaded solid lipid nanoparticles for oral administration

Oral delivery of biological drugs is the pharmaceutical challenge of improving administration of biomolecules by overcoming the harsh conditions of the gastrointestinal tract (GIT). Solid lipid nanoparticles (SLNs) are among the most promising carrier candidates to encapsulate biomolecules and deliver them through the GIT tract. In this project, SLNs were manufactured through microfluidics using an ethanolic solution of lipid mixture 1:3 (mol/mol) HSPC/cholesterol, and aqueous phase of 1:12 molar ratio exenatide/DOTAP in 1 mM HEPES buffer at pH=8, so that the peptide feed was 10% (w/w) with respect to the SLN mass. Through hydrophobic ion pairing (HIP) method, DOTAP/ exenatide assemblies are guided in the bulk of the lipidic matrix during the microfluidic process, improving peptide’s loading efficiency. Exenatide loaded SLNs were characterised in size, PDI and z-potential by DLS, while loading efficiency and capacity were determined by RP-HPLC analysis. The SLN melting temperature was assessed by DSC. To promote absorption through the intestinal barrier, mucodiffusivity requires that SLN possess neutral or anionic surface, thus shielding of the cationic surface charge was explored using polyanionic or amphiphilic polymers. Coating with anionic polymers through electrostatic interaction exploitation was first performed with tripolyphosphate (TPP) adsorption, which was not tightly associated to nanoparticles’ surface. Subsequently, electrostatic association was used to coat the cationic nanoparticles using hydrophilic anionic homopolymers, namely poly-a-[50]-glutamic acid (PGA) and a block copolymer PEG2kDa-poly-a-[10]-glutamic acid (PEG-PGA). Coating the cationic SLNs with increasing polymer/SLN (w/w) ratios, monitored by DLS, resulted in the decrease of the naked SLNs z-potential, and the generation of stable and highly anionic coated SLNs. The threshold of PGA or PEG-PGA/SLN ratio that allowed for well dispersed particle with size comparable to the naked ones was identified.The third SLN coating strategy used an amphiphilic material, DSPE-mPEG(2 kDa), associated to the SLN surface by associating the DSPE anchor within the surface matrix of preformed SLNs. Two selected formulations, 10%- and 30%- DSPE-mPEG/SLN (w/w) ratio were characterised and resulted in charge lower than 30 mV and an almost neutral zeta potential with respect to that of the naked SLN, respectively. FRAP method was used to investigate mucodiffusivity of naked and PEGylated (10%, 30%) nanoparticles showing that coating with DSPE-PEG restores SLN mobility. Release profile of exenatide from the SLNs was studied in SIF and in physiological buffer (PBS); peptide was quantified by RP-HPLC. Subsequently, colloidal stability of SLN in GIT mimicking environments was performed including simulated gastric fluid (SGF), SGF with pepsin (SGF wp), SIF and SIF with trypsin (SIF wt), pancreatic lipase, and bile extract. PEGylated SLNs were stable in most of conditions (SGF, SIF, SGF wp, SGF wt) while naked SLNs underwent size increase in SGF wp, SIF and SIF wt. Both the nude and the PEGylated (10, 30%) nanoparticles were subjected to mild lipolysis after 4 h of incubations, as well as emulsification in presence of bile. Finally, the SLN encapsulated peptide stability was assessed upon incubation of naked of PEGylated particles (10%, 30%) in SGF wp and SIF wt. All nanoparticles offered protection from degradation with a different degree between the tested formulations, with PEGylated (10%, 30%) outperforming the nude ones. However, the 10%DSPE-PEG/SLN (w/w) ratio formulation offered a markedly increased protection than the 30%%DSPE-PEG/SLN (w/w) ratio formulation. This was probably due to the presence in the 10%DSPE-PEG/SLN (w/w) ratio formulation of a brush like PEG layer with marked obstruction of the proteases, while in the 30% counterpart, the mushroom/brush conformation of PEG layer may allow infiltration of the proteases which will digest the surface peptide