Gene therapy can potentially treat a wide range of diseases ranging from inherited diseases to cancer. The successful use of nucleic acids to treat genetic diseases is limited by rapid capture and degradation of the nanoparticle by Kupffer cells in the liver. Scavenger receptors on the cell surface, capture both viral and non-viral nanoparticles leading to reduced efficacy. PEG-peptides were found to inhibit scavenger receptors on the surface of Kupffer cells by forming albumin nanoparticles when intravenously dosed. This work explores the development of potent, low-molecular weight PEG-peptide inhibitors. In order to study the in vivo activity of the nanoparticle, an in vivo assay was developed to directly assess the potency of inhibition. High molecular weight polylysine peptides (33.5 kDa) inhibited liver uptake with an IC50 of 18 μM. Incorporation of four leucine residues, to improve albumin binding, allowed for a decrease in PEG molecular weight and number of lysine residues, resulting in PEG5kda-Cys-Tyr-Lys-(Leu-Lys4)3-Leu-Lys (7.4 kDa) that inhibited scavenger receptors with an IC50 = 20 μM. Further decrease in the PEG molecular weight resulted in the discovery of PEG2kDa- Cys-Tyr- (Leu-Lys4)3-Leu-Lys (4.4 kDa) with potency of 3 μM. The increase in potency could be attributed to a decrease in the zeta potential of the albumin nanoparticle resulting in more efficient scavenger receptor mediated uptake. Co- administration of PEG2kDa- Cys-Tyr-(Leu-Lys4)3-Leu-Lys with a stable PEGylated polyacridine DNA polyplex resulted in inhibition of rapid polyplex uptake by the liver with an IC50 = 11 μM. Other properties including spatial distribution of leucine, hydrophobicity and peptide length were also explored to determine their effect on liver uptake. Hydrophobic peptides resulted in the formation of micelles which were inactive as scavenger receptor inhibitors and exhibited increased liver uptake upon dose escalation. Reduction in the peptide length resulted in peptides that were not captured by the liver. Inhibition scavenger receptors has the potential to improve the efficacy of viral and non-viral nanoparticles. The findings of this work provide a framework for the development of PEG-peptide inhibitors capable of blocking live uptake of viral and non-viral nanoparticles.

In pharmaceutical development cell cultures are used as in vitro models to evaluate the function of drug candidates. In such research it is vital to have models that resemble the in vivo environment to get reliable results. In 3D models with hydrogels ECM like scaffolds are supporting the cells in a more in vivo like environment than flat 2D cultures. In this project PEG-peptide based hydrogels with cell binding RGD incorporated on one PEG-peptide type has been evaluated for culturing of HepG2 cells. Structure and viscoelastic properties were evaluated with techniques like circular dichroism spectroscopy, dynamic light scattering and rheology. Sterilisation impact was also evaluated for PEG-peptides. For cell culturing, observations in light microscope and evaluation with Live/Dead assay and albumin assay were performed. A few companies were interviewed regarding 3D culturing and interest in mechanically tuneable hydrogels. The HepG2 cells grows and forms spherical clusters in the 3D environment with hydrogels, percentage of RGD seems to not impact cell adhesion, growth or albumin secretion. UV irradiation was the most suitable sterilisation method for gel components. The most rigid gel combination formed had storage modulus of around 230 Pa. Mechanically tuneable hydrogels is interesting for the industry. The PEG-peptide based gels are suitable tor growing cells but too soft to closely resemble the in vivo rigidity of hepatocytes.