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Identifying Parameters That Impact Endosomal Uptake and Endosomal Release of a Model Cell Delivery Reagent
Abstract
Intracellular delivery agents are useful tools to introduce cargos into mammalian cells. Delivered cargos can be utilized to study biomolecules in the context of mammalian cells, to trigger a biological activity of mammalian cells, or to genetically modify cells. One major bottleneck that plagues intracellular delivery is that the sources that cause inefficient cytosolic entry of delivery agents are not easily identifiable. Hence, the parameters that impact delivery efficiency must first be determined to design better delivery systems. The success of the delivery system is dependent on two main parts, the context in which the delivery agent is introduced and the chemical properties of the delivery agent. To study the environment in that the delivery agent is introduced, the studies herein assessed the impact of cell culture protocols and different tissue culture lines on the uptake of a delivery agent TMR-TAT. Different protocols examining the influence of cell dissociation, media components, and temperature yielded uptake as low as 2.8 median fluorescence intensity (MFI) and as high as 37.5 MFI (a range of approximately 13-fold). Additionally, TMR-TAT exhibited broad uptake efficiencies among 6 different cell lines in a range of approximately 10-fold. To quantify the extent to which alterations to a model delivery agent dfTAT impacts its delivery efficiency, the influence of attachment of different sizes of cargos (ranging from 1.4 kDa to 29 kDa) to a model delivery agent dfTAT was established., Attachment of peptide cargos (1.4 and 2.8 kDa additions respectively) to dfTAT (4.1 kDa) did not impact delivery efficiency. In contrast, the attachment of snoopcatcher (14.5 kDa) to dfTAT-ST (5.5 kDa) resulted in a 60% decrease in delivery efficiency. Additionally, the attachment of two snoopcatcher proteins to dfTAT-dST (6.9 kDa) nearly abolished delivery (>5% delivery). The information gathered here can potentially be utilized to design next generation TAT peptide-based delivery systems by modifying protocols, selecting optimal cell types, and developing strategies to enhance TAT-cargo conjugate systems. The approaches established herein may also be applied to other delivery agents to identify parameters that impact delivery efficiency.
Subject
Cell penetrating peptidesendocytosis
intracellular delivery
bioconjugation
tissue culture protocols.
Citation
Diaz, Joshua D (2023). Identifying Parameters That Impact Endosomal Uptake and Endosomal Release of a Model Cell Delivery Reagent. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /199865.