Browsing by Subject "peptides"

Oncolytic adenoviruses are a new cancer treatment platform which aims to eliminate cancer through direct lysis of cancer cells by viral replication and the activation of the immune system by the release of tumor antigens upon oncolysis. In the PeptiCRAd technology, the activation of an anti-cancer immune response is enhanced by the addition of poly-lysine modified cancer peptides, where the antigen presentation to the immune system is improved in comparison to plain oncolytic viruses. PeptiCRAd complexes have been assumed to form solely by electrostatic interactions, but the thermodynamic profiles and mechanisms involved in the complexation have not been previously addressed. Thus, by adding isothermal titration calorimetry as part of the analysis repertoire provides valuable information of the characteristics of PeptiCRAd complexes. In this study, the applicability of isothermal titration calorimetry in PeptiCRAd complexation analyses was evaluated based on initial peptide-to-virus and virus-to-peptide titrations, and a method of analysis was created for the thermodynamics of the interactions of the complex. Optimization of the experimental method (i.e., titration protocol) and the data analysis (i.e., calculation models) remains inconclusive for quantitative analysis as data obtained from the measurements was mainly of bad quality, thus requiring further optimization to obtain reliable data. However, using surface plasmon resonance as an already established method for poly-lysine peptide-virus interaction studies gave robust data and can be used as a base or guideline to further develop isothermal titration calorimetry analyses for characterizing PeptiCRAd complexes. Although isothermal titration calorimetry measurements were unsuccessful for quantification purposes, it was possible to qualitate the mechanisms of PeptiCRAd complexation for four different peptides with fair confidence. The peptides showed low heats of binding, and positive and negative cooperative binding in ionic and non-ionic solutions, respectively. Based on this, the binding of peptides in PeptiCRAd complexes was determined to be driven by hydrophobic inter-peptide interactions on the virus surface, although an electrostatic attraction is indeed present at the virus-peptide interface, initiating the binding event. Also, improvements to the titration protocol for PeptiCRAd analyses with isothermal titration calorimetry are suggested for further optimizations in the future to conclusively determine the applicability of the isothermal titration calorimetry technique for characterizing peptide-virus interactions of PeptiCRAd complexes.

Liposomes are biocompatible spherical nanosized vesicles consisting of hydrophobic phospholipid bilayer encasing an aqueous core. They can be utilized as drug carriers by either encapsulating molecules inside the core or embedding them in the bilayer accordingly to achieve numerous advantages such as prevention of rapid clearance and reduction of adverse effects as systemic exposure is reduced. Despite the marked efforts in designing the liposomes to improve therapeutic outcomes, only limited drug concentrations are achieved at the target sites such as in solid tumors. Stimuli-responsive liposomes could be applied as potential delivery systems to achieve spatiotemporally controlled drug delivery, i.e., the drug release could be pinpointed and restrained to the target site. In this thesis, the objective was to study the light-activated indocyanine green (ICG) liposomes as nanocarriers for peptide-based anti-tumor agents. The physicochemical characteristics, stability and functionality of the prepared liposomes were determined alongside optimizing the formulation as needed and utilizing different model peptides as encapsulated compounds. Additionally, the peptide stability during near-infrared (NIR) light illumination and the effects of the anti-angiogenic model peptides in vitro were investigated. The stability of the liposomes was assessed by monitoring the size of the liposomes, intactness of ICG, and passive leakage of the peptides over time, and by determining the phase transition temperatures of the different formulations. The liposomes remained adequately stable in different relevant conditions, and the observed phase transition temperatures did not indicate the lipid bilayer becoming permeable in physiological temperatures. However, the rate of passive leakage was rather high in all formulations, although with stiffer lipid bilayer in the “rigid” formulation, the unintended release was able to be decreased slightly in comparison to the other formulations. On the other hand, light-triggered release upon illuminating the liposomes remained considerably low in all formulations. The intactness of peptides seemed to not be impacted by the illumination. Also, no cytotoxic effects were observed after exposing human umbilical vein endothelial cells (HUVEC) to the peptides. The final “rigid” formulation showed the best functionality out of those included in the studies. It remains to be investigated whether the formulation could be improved further for optimal functionality and stability, and to what degree do the properties of the cargo molecule affect the performance of the liposomes.