| Abstrakt: |
Delivery tools, including cell-penetrating peptides (CPPs), are often inefficient due to a combination of poor endocytosis and endosomal escape. Aspects that impact the delivery of CPPs are typically characterized using tissue culture models. One problem of using cell culture is that cell culture protocols have the potential to contribute to endosomal uptake and endosomal release of CPPs. Hence, a systematic study to identify which aspects of cell culturing techniques impact the endocytic uptake of a typical CPP, the TMR-TAT peptide (peptide sequence derived from HIV1-TAT with the N-terminus labeled with tetramethylrhodamine), was conducted. Aspects of cell culturing protocols previously found to generally modulate endocytosis, such as cell density, washing steps, and cell aging, did not affect TMR-TAT endocytosis. In contrast, cell dissociation methods, media, temperature, serum starvation, and media composition all contributed to changes in uptake. To establish a range of endocytosis achievable by different cell culture protocols, TMR-TAT uptake was compared among protocols. These protocols led to changes in uptake of more than 13-fold, indicating that differences in cell culturing techniques have a cumulative effect on CPP uptake. Taken together this study highlights how different protocols can influence the amount of endocytic uptake of TMR-TAT. Additionally, parameters that can be exploited to improve CPP accumulation in endosomes were identified. The protocols identified herein have the potential to be paired with other delivery enhancing strategies to improve overall delivery efficiency of CPPs. [ABSTRACT FROM AUTHOR] |
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