Overcoming biological barriers by lipid-based nanocarriers

Oligonucleotides (ONs) have recently been studied as therapeutic agents for various pathologies such as genetic disorders, and cancer. A possible delivery system for ONs can be the formulation of lipoplexes, which consists of complexes between cationic liposomes and ONs. Within this thesis project, lipoplexes were formulated for the delivery of ONs and were prepared using an oligocationic enhancer (OCE). OCE provides the advantages of condensing the ONs, allows a high loading degree, and is a cell penetration enhancer. 4% of OCE in lipid mixture was selected for providing high surface charge and high membrane association efficiency. The effect of the lipid saturation on the lipoplexes was investigated using saturated phospholipid HSPC or unsaturated phospholipid EPC. Even though EPC-based lipoplexes have shown similar colloidal characteristics, they were excluded from the study for their fast-release profile. For the determination of the optimum N/P ratio, the lipoplexes HSPC-based were generated at decreasing N/P ratios from 10:1 to 1:1 and a 10:1 N/P ratio was selected for providing small particle size, low polydispersity and high loading efficiency. Lipoplexes have shown great stability up to 4 IU/mL heparin concentration and hemocompatibility. Cytotoxicity studies showed that lipoplexes induced negligible toxicity. Cellular association studies showed that the OCE increases the association of the lipoplexes. Silencing efficiency studies showed the inclusion of the fusogenic lipid DOPE significantly increases the eGFP silencing. The silencing efficiency of the 1:1 HD lipoplexes was also shown by imaging with confocal microscopy. Lipoplexes showed a limited colocalization with the acidic compartments suggesting that DOPE is triggering the endosomal escape of the lipoplexes resulting in high silencing efficiency. Diabetes mellitus (DM) is a disorder that can be caused by insulin resistance in the peripheral tissues (Type 2, T2DM). One of the options for T2DM treatment is the use of GLP-1 analog exenatide (EXEN). In this project, we aimed at contributing to the oral delivery strategies of therapeutic peptides by developing EXEN-loaded solid lipid nanocarriers by microfluidic technology by the hydrophobic ion pairing (HIP) method. HIP between EXEN and cationic lipid DOTAP was shown by the isothermal titration calorimetry. The process parameters were identified as 3:1 FRR and 12 mL/min TFR for the small (<100 nm) and homogeneous size distribution (PDI < 0.2). SLNs produced by processing 5-15% (w/w) EXEN/total lipid feed ratio and 10% (w/w) EXEN/total lipid feed ratio and 12:1 DOTAP/EXEN (mol%) showed the most desirable features. The release of the EXEN from SLNs in SIF at pH 6.8 and in PBS at pH 7.4 showed burst release was observed in the first 8 hours followed by a prolonged release of 10 days. SLNs showed high colloidal stability of SLNs in SIF and PBS. The positive charges of SLNs were masked by decoration with PEG and 10 and 30% w/w ratios were selected as mean and high PEG density. TEM imaging showed a spherical morphology of SLN. FRAP analysis was conducted to investigate the SLNs' mobility in the mucin and the results showed that the diffusion of the SLNs can be modulated by PEG coating. SLNs were converted into lyophilized powder for loading in capsules. A mixture of trehalose and mannitol (1:1 w/w) at 2% (w/v) concentration was selected for ensuring the redispersion of the SLNs. The stability of EXEN throughout the formulation process was shown by CD and ESI-TOF MS. The biocompatibility study SLNs with Caco-2 cells showed a safe profile. The cellular association of the SLNs showed over 50% association efficiency of the not PEGylated SLNs while the PEGylation association was decreased. Enteric coating of capsules was done with Eudragit L100 polymer: showing high integrity for 2 h in HCl pH 1.2 and in SIF a rapid disintegration of the capsule and release of over 80% of the content in 1 hour was observed.