The immune system is responsible for maintaining health, as it is able to fight off harmful pathogens and detect one's own altered cells. When the altered cells proliferate and spread uncontrollably, cancer can develop. One of the most aggressive cancer types is pancreatic cancer, the most common form being pancreatic ductal adenocarcinoma (PDAC), which is mostly caused by the accumulation of mutations in the KRAS gene. Low immunogenicity and a resistant tumor microenvironment contribute to a poor prognosis. Nanoparticle-based systems in the form of nanovaccines for immunogen (and adjuvant) delivery show promising results in improving treatment outcomes.
Within this project, polymeric nanoparticles were formulated by a double emulsion solvent evaporation method, using different polymers; polycaprolactone (PCL), polycaprolactone-polyethylene glycol copolymer (PCL-PEG) and polyoxazoline (POx). We have produced nanoparticles without bioactive molecules, and the ones with antigenic peptides (KRAS G12D MHC I/II), and/or hydroxypropyl-β-cyclodextrin (HP-β-CD). We evaluated physico-chemical properties, such as nanoparticle size distribution and zeta potential. Nanoparticle average diameter was below 200 nm with a polydispersity index ranging from 0 to 0.2. Zeta potential was near-neutral. By indirect quantification, we found that we achieved good antigen incorporation with 80% entrapment efficiency and a loading capacity of 40 µg/mg. To assess the potential toxicity of the nanoparticles, we performed in vitro cell viability assays using the MTT assay and the Live/Dead kit. For this, we used JAWSII, an immature dendritic cell line, and the KPC pancreatic tumor cell line. The Live/Dead kit assay showed a high viability of the JAWSII cell line, above 80% (relative to untreated control cells), for all nanoparticles at all time points tested, with no significant differences when HP-β-CD was added. However, the MTT assay showed that HP-β-CD induced proliferation of the JAWSII cell line, while the lowest viability, down to 50%, was detected after treatment with "empty" nanoparticles. In contrast, we did not observe any effect on the viability of the KPC cell line when antigenic peptides or cyclodextrin were incorporated into nanoparticles. Furthermore, we conducted an in vitro internalization assay of the nanoparticles by the JAWSII cells, using flow cytometry. The presence of HP-β-CD did not promote nanoparticle uptake. Based on the results obtained, it can be concluded that successful formulation and physicochemical evaluation of the nanoparticles was achieved. Cell viability and internalization assays provided insights into the immunomodulatory potential of the nanoparticles, which may contribute to the understanding of their efficacy as a nanovaccines for the treatment of PDAC.
|
