Cell-penetrating peptides for enhanced oral delivery of nanoformulations

Introduction: Oral drug delivery systems such as polymeric nanoparticles are used to improve therapies that utilize biomacromolecules like proteins and peptides. Surface modifications of polymeric nanoparticles play a crucial role in the interactions with the intestinal epithelium. Cell-penetrating peptides (CPPs) are short cationic amino acid sequences that can be utilized to enhance interactions between polymeric nanoparticles and cells. In this thesis, surface-modified polymeric nanoparticles are prepared using a nanoprecipitation method and a zero-length crosslinking reaction for the covalent conjugation of CPPs to polymeric nanoparticles. Three CPPs with a distinct architecture, namely the short RRH, the long linear TAT and the branched bTAT were exploited. Further, the nanoparticles were characterised and the influence of the CPP architecture on cellular uptake was investigated. Methods: A bulk nanoprecipitation and a microfluidics method were compared for the formulation of uniform poly-lactic-co-glycolic acid (PLGA) nanoparticles using a design of experiments study. CPP-tagged PLGA nanoparticles were formulated using a post-microfluidics and an in situ microfluidics conjugation approach developed for the first time. The physiochemical characteristics and morphology of PLGA and CPP-tagged PLGA nanoparticles were analysed with dynamic light scattering, laser Doppler electrophoresis, Fourier-transform infrared spectroscopy and transmission electron microscopy (TEM). The distribution of CPPs on PLGA nanoparticles was further elucidated with small angle X-ray scattering (SAXS) after gold labelling of the CPP-tagged PLGA nanoparticles. PLGA and CPP-tagged PLGA nanoparticles were prepared using a fluorophore-labelled PLGA for in vitro cell culture studies. The cell toxicity and interactions of the fluorophore-labelled PLGA and CPP-tagged PLGA nanoparticles with HeLa and Caco-2 cells were investigated using flow cytometry and confocal laser scanning microscopy. Results: PLGA nanoparticles intended for oral drug delivery and formulated with microfluidics showed a size of 151.2 ± 1.2 nm (PDI 0.149 ± 0.014) and had superior size characteristics in comparison to the bulk nanoprecipitation method resulting in PLGA nanoparticles with a size of 184.0 ± 3.9 nm (PDI 0.110 ± 0.007). The covalent conjugation of CPPs with different architectures tuned the surface charge of CPP-tagged PLGA nanoparticles from negative to slightly positive (-24 to +5 mV). This trend in change of surface charge was observed for both preparation methods, the post-microfluidics and the in situ microfluidics conjugation approach. After analysis with TEM and SAXS, it was found that the distribution of CPPs on PLGA nanoparticles depends on the preparation approach. The in situ microfluidics conjugation approach showed a distribution of the CPPs throughout the PLGA nanoparticles, whereas the post-microfluidics conjugation approach indicated a surface arrangement of the CPPs on the PLGA nanoparticle surface. In vitro cell culture studies using HeLa and Caco-2 cells revealed association rather than uptake of the CPP-tagged PLGA nanoparticles. Conclusion: Microfluidics and CPPs of different architecture were successfully utilized for the formulation of surface-modified polymeric nanoparticles with a tuneable surface charge. For nanoparticle-cell interactions to occur e.g. at the nano-bio interface, the surface charge of nanoparticles plays a crucial role. Further investigation of nanoparticle-cell interactions can aid to gain a better understanding of how the well-characterised CPP-tagged PLGA nanoparticles presented in this thesis can influence cellular uptake