Targeted delivery of nucleic acids to cancer associated fibroblast using lipid nanoparticles

Tumor microenvironment is very complex structure made our extracellular matrix and variety of cells. One of the more prominent cell types are cancer associated fibroblast (CAFs). They are involved in variety of processes that promote tumor growth and development. For years tumors have been treated with traditional therapies such as radiotherapy or chemotherapy. However, they all have their own drawback and lead to reduced quality of life for patients. That is why alternative treatment are gaining more popularity. One of them are nucleic acid therapeutics. However, they also have their own drawbacks like the need to be delivered using a vesicle that will be able to protect them and assist in cell membrane penetration. The purpose of this study was to produce lipid nanoparticles (LNPs) that will be able to encapsulate nucleic acid for targeted delivery to cancer associated fibroblasts. Ionizable lipid Dlin-MC3-DMA (MC3) was used to encapsulate nucleic acids. Various factors were taken into consideration during the LNPs production process. Those include flow rate, lipid:RNA volume ratio and dye usage. Moreover, conjugation and purification were also adjusted to further optimize the process. AV3 molar excess, TCEP usage and purification methods were shown to impact the size of the LNPs. All tested conditions resulted in lipid nanoparticles capable of nucleic acid encapsulation. To proceed with in vitro studies, pancreatic stele cells (PSCs) treated with transforming growth factor beta (TGF-β) were tested to see if they were activated and had overexpressed integrin alpha-5 (ITGα5). qPCR proved activation of PSCs with TGF-β. Additionally immunostaining and qPCR proved overexpression of ITGα5 on treated cells. Uptake studies confirmed that AV3 conjugated LNPs are capable of targeting activated PSCs. Transfection studies demonstrated targeting and nucleic acid delivery by LNPs-AV3.