Structural, hydrodynamic and thermodynamic analysis of a novel drug delivery vector ELP[V5G3A2-150] in PBS, with preliminary analyses for applying quantitative hydrodynamic analyses in serum.

摘要

Recent years have seen a dramatic acceleration in the development of cancer therapeutics. They are increasingly designed to target processes or features that are either unique or up-regulated in cancer cells, thereby providing selectivity towards cancerous cells. However, the majority of therapeutics still cause unacceptable systemic toxicity to non-cancerous cells. Elastin-like polypeptides (ELPs) are currently being developed components of a drug delivery vector that will allow for physical targeting of systemically delivered drugs. ELPs are derived from the endogenous protein elastin and exhibit a structural change at a transition temperature (TT). Below the TT, ELPs are soluble in aqueous solution, but above the TT, structurally transition and aggregate. The hypothesis is that an ELP with a precisely engineered TT that is above physiological temperature (TP) but below the temperature achievable in an artificially heated tumor (TH) can be delivered systemically and will aggregate at the site of the tumor, thereby targeting any covalently attached therapeutic. This hypothesis has been tested in vitro and in vivo and very promising results have been obtained.;In this study, ELP has been examined using a variety of complementary biophysical techniques as a means of understanding and predicting its behavior as a drug delivery vector. Consistent with previous studies, turbidity studies revealed that the aggregation is both reversible and repeatable, and that the TT is inversely related to the logarithm of the ELP concentration. Dynamic light scattering, circular dichroism, and analytical ultracentrifugation revealed that at low temperatures (5°C), ELP is soluble and adopts an extended, disordered conformation. As the temperature is increased, ELP experiences increasing amounts of weak association that correspond to a decrease in percent disordered structure and corresponding increase in percent β-conformation. Above the TT ELP does not aggregate fully, but leaves a critical concentration of soluble protein that decreases with increasing temperature. This evidence, combined with the logarithmic dependence of the TT on the concentration and the weak association that increases with temperature, led us to postulate that the TT is actually a measure of ELP's solubility constant. This has great significance for ELP as a drug delivery vector as it suggests that there will always be a constant, critical concentration of ELP circulating systemically.;Since the development of ELP it has been subsequently modified with the addition of cell penetrating peptides to increase tumor uptake. The effect of adding cell penetrating peptides and changing lysine residues to cysteines for drug attachment were investigated. We hypothesized that increasing the number of charged residues would increase the solubility of ELP. This would be observed as an increase in the TT. All constructs retained the ability to reversibly and repeatedly aggregate at high temperatures. Interestingly, lowering the pI by replacing the only charged residue, lysine, with a cysteine raised the TT. Consistent with this observation, the addition of very basic CPPs lowered the TT of ELP. This was opposite to the anticipated effect. We believe this may be caused by the formation of micelles due to the asymmetric distribution of charges. Otherwise the biophysical properties were unaffected by the addition of CPPs. These results suggest that these CPPs are suitable for conjugation to ELP as a means of increasing drug delivery efficacy.;When ELP was sedimented in serum the Johnston-Ogston (J-O) effect was observed. The J-O effect is a classical anomaly in sedimentation history that describes the apparent increase in concentration of a component in the presence of a faster sedimenting component. Due to the complexity of serum, we chose to reduce serum to its two most ubiquitous components, albumin (BSA) and gamma-globulins (IgG). We carefully investigated the J-O effect using advances in sedimentation hardware, the fluorescence detection system (AVIV-FDS), and advances in analytical ultracentrifugation software analysis, global direct boundary fitting as implemented in Sedanal. We measured the presence of cross-term hydrodynamic non-ideality. This phenomenon has been addressed by adding a matrix of cross-term non-ideality terms into Sedanal. We were able to fit data of ELP sedimenting in both BSA and IgG, however we were still unable to fit the data of ELP sedimenting in serum. This suggests that there is additional complexity in serum that is unaccounted for in the fitting parameters. Current investigations involve determining how lipids, which float during sedimentation, will affect the sedimentation parameters.