Assistant Professor Villanova University, United States
Introduction:: Non-viral delivery vehicles are a promising method to transport cargo such as drugs, nucleic acids, and other biological factors into cells, however, it is important to understand what occurs once the vehicles are endocytosed to maximize therapeutic activity. Endosomal escape is a critical part of the delivery process and could be the determining factor in the success of a particular treatment [1]. If the vehicle is unable to escape the endosome, then the important cargo that is being delivered will not be able to carry out its intended role. Therefore, accurately quantifying and learning how to maximize endosomal escape once the delivery vehicle has entered the cell is extremely important in the success of the treatment. The purpose of this study is to investigate the endosomal escape of a library of different poly(amine-co-ester) (PACE) nanoparticles (NPs) with various surface and chemical properties to define a set of rules for endosomal escape of solid polymer NPs in endothelial cells (ECs). PACE is a cationic polymer that can strongly complex with nucleic acids and aid in cell infiltration, but with low charge densities that reduce toxicity compared to other polymers, such as PEI and PBAE [2]. This work is done using several different methods to most accurately identify the pathway and which PACE vehicles are escaping the endosome.
Materials and Methods:: Human umbilical vein endothelial cells (HUVECs) with stably transfected fluorescent early endosomes (GFP-RabA5) and lysosomes (RFP-LAMP1) were developed for this work, in order to quantitatively track fluorescent NPs overtime within a single cell population. NPs from different types of PACE polymers including PEGylation, acidic end-groups, and varying ratios of monomers effecting the hydrophobicity and charge, containing CY5-tagged-dsDNA were incubated with HUVECs. Time and dose related variables were assessed for the effect on cell internalization. The colocalization of the fluorescent dsDNA with the endosomes and lysosomes were measured using fluorescence microscopy (EVOS) to investigate the cellular uptake of the NPs and dsDNA into the cell as well as the dynamics of the endosomal pathway. Chloroquine was also added to investigate the effects of prevention of endosomal maturation.
Results, Conclusions, and Discussions:: A portion of the NPs tested that have not accomplished endosomal escape can be seen in the graph above. The results have shown a decrease in colocalization between the dsDNA and the endosomes (18% to 12%) and an increase in colocalization between the dsDNA and the lysosomes (9% to 20%) around the 24-hour mark as compared to the four-hour mark. About 70% of the NP area is not in an endosome or lysosome, so it is possible that those NPs are making their way into the cytosol, considering the cells are washed many times with PBS and media before imaging. The endosomes containing the remainder of the NPs that are not achieving endosomal escape seem to be maturing into lysosomes. At day four, the results have shown a consistent a decrease in the overlap of the dsDNA with both vesicle types, suggesting degradation of the NP and its cargo, dsDNA. This has also been confirmed by the use of pharmaceutical inhibitors. Preliminary data suggests that adding additional charged groups to the surface may reduce the NPs that are transitioned to lysosomes and enhance endosomal escape.
These data suggest that these methods used can appropriately measure colocalization of endocytic vesicles with the PACE NPs containing fluorescent dsDNA. This will be used to track the endocytic pathway of a library of PACE NPs to determine which can escape the endosomes and warrant further investigation as prospects for nucleic acid delivery as a treatment for inflammation in endothelial cells.
Acknowledgements (Optional): :
References (Optional): : 1. Xu, E., W.M. Saltzman, and A.S. Piotrowski-Daspit, Escaping the endosome: assessing cellular trafficking mechanisms of non-viral vehicles. J Control Release, 2021. 335: p. 465-480.
2. Piotrowski-Daspit, A.S., et al., Polymeric vehicles for nucleic acid delivery. Adv Drug Deliv Rev, 2020. 156: p. 119-132.
