This study describes the design and development of multifuctional poly(amidoamine)s (PAAs) capable to form self-assembled nanocomplexes with peptides and proteins, as functional bioresponsive vectors for protein delivery to targeting cells in vitro and in vivo. The representative examples of this novel family of water-soluble PAAs have very promising properties to function as vectors for highly potent and non-toxic intracellular protein delivery. The synthesis procedure of these polymers is compatible with the use of a variety of functionalized amine monomers, allowing large variation in the structure of the polymers, and tuning of the chemical and biophysical properties that are of significance for efficient protein delivery. We have designed PAAs able to form nanosized complexes at neutral pH by a self-assembly process with model proteins, such as β-galactosidase, lysozyme, human serum albumin, ovalbumin, p24 and human insulin, and a model peptide for brain therapy. These nanocomplexes are stable at physiological pH and efficiently protect protein integrity (i.e. no denaturation occurs). Upon acidification of the solution after endosomal uptake, the pH decrease and consequently the change of charge lead to decreased protein-polymer interaction and thus a tendency for the nanoparticles to destabilize. Moreover, for nanocomplexes made from PAAs containing disulfide linkages in their polymeric backbone (SS-PAAs), fast disintegration of the complexes and release of the protein payload is observed in reductive solution, mimicking the intracellular environment. Profection experiments showed that proteins encapsulated in PAA nanoparticles were successfully internalized into the cells with remaining activity and excellent cell-viability. In addition of providing intracellular bioreduction, the presence of the disulfide groups also increased the mucoadhesive properties of the nanoparticles. In the presence of mucus secreting cells, we showed that the SS-PAA-based nanocomplexes were capable to successfully internalize protein into the cell cytosol with a much higher extent than nanoparticles based on PAAs lacking disulfide linkages. The results described in this thesis indicate that PAAs should create opportunities for novel applications in protein delivery for oral and nasal administration, as well as delivery to the brain.
|24 Jun 2011
|Place of Publication
|Published - 24 Jun 2011
- EC Grant Agreement nr.: FP6 IP NANOBIOPHARMACEUTICS