Development of solid lipid nanoparticles by microfluidic technique for oral delivery of peptides

This thesis project was aimed to develop solid lipid nanoparticles (SLNs) based formulation for the oral delivery of a model peptide. In order to obtain a reproducible and scalable formulation process, microfluidic technology (Nanoassembler® Benchtop) was exploited to prepare SLNs. SLNs were formulated by processing an organic and an aqueous phase with a total flow rate (TFR) of 12 mL/min and an aqueous/organic flow rate ratio of 3:1. The lipid mixture was composed of hydrogenated phosphatidylcholine from soybean (HSPC) and cholesterol in ethanol, while in the aqueous phase peptide has been precomplexed with a cationic phospholipid, DOTAP, under basic pH conditions to provide a negative net charge of peptide. The hydrophobic ion pairing (HIP) technique was employed to increase the lipophilicity of the peptide thus promoting its encapsulation in the lipid matrix of the SLNs, in virtue of the electrostatic interaction between the peptide and DOTAP. Preliminary studies using a 5% (w/w) peptide/lipid feed ratio were performed to select the buffer concentration that ensures the highest loading, small and homogenous SLNs. The formulations processed with 1 mM HEPES buffer at pH 8 resulted in particle size of about 120 nm, and PDI about 0.13. A library of SLNs was produced by processing 5, 10 and 15% (w/w) peptide/lipid feed ratio. Each set of formulations was produced with pre-complexation of the peptide at increasing DOTAP/peptide molar ratio, namely 0:1, 2:1, 6:1, 12:1 and 18:1. SLNs obtained with 10% (w/w) peptide/lipid feed ratio and 12:1 DOTAP/peptide molar ratio showed the most desirable pharmaceutical features in terms of size, PDI, and loading. Indeed, the nanoparticles had a particle size of 123.7 ± 2.5 nm, a PDI of 0.09 ± 0.01, a loading efficacy of 94.3 ± 0.4% and a loading capacity of 9.6 ± 0.2%. Therefore, this formulation was selected for further characterization and formulation. To gain detailed physicochemical information about the interaction between peptide and DOTAP, isothermal titration calorimetry (ITC) was exploited using 10% (w/w) DOTAP containing liposomes as a model. At pH 8 the negatively charged peptide in the sample cell was injected with DOTAP-liposome generating an exothermic enthalpy variation whose intensity decreased over the sequential injections. The control test, performed at pH 3.5, where the peptide is positively charged as the liposomes, showed no enthalpic changes confirming the electrostatic interaction between DOTAP and the peptide. The release of the peptide from SLNs was investigated in simulated intestinal fluid (SIF) at pH 6.8 and in phosphate-buffered saline (PBS) at pH 7.4 to mimic the intestinal lumen and the extracellular conditions, respectively. Under both conditions, a burst release was observed in the first 8 hours followed by a prolonged release of 10 days in SIF and 14 days in PBS. SLNs were then converted into lyophilized powder for loading in enteric-coated capsules. Trehalose was identified as the preferential cryoprotectant to maintain the quality of nanoparticles. Increasing concentrations of trehalose were tested and 1% (w/v) was selected for being the lowest concentration of this excipient that allow the redispersion of particles without aggregation (size = 139.0 ± 3.9 nm, PDI = 0.19 ± 0.01). Preliminary investigations were made to evaluate the effectiveness of the enteric-coating on hard gelatine capsules, to avoid their premature breaking in the gastric environment. Enteric coating composed of Eudragit L100, permitted the maintenance of capsule integrity at pH 1.2, which mimics gastric conditions. Instead, when the capsules were transferred in a simulated intestinal fluid at pH 7.4, they rapidly disintegrated to allow the release of their content.